Exosome-based cancer assays

ABSTRACT

The technology described herein is directed to methods of treating and diagnosing cancer, e.g, by measuring the expression of certain genes in exosomes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application Nos. 63/171,689 filed Apr. 7, 2021 and63/277,766 filed No. 10, 2021, the contents of which are incorporatedherein by reference in their entirety.

GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos.CA222170 and CA24300 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 5, 2022, isnamed 701586-099990USPT_SL.txt and is 1,110,069 bytes in size.

TECHNICAL FIELD

The technology described herein relates to methods of treating anddiagnosing cancer.

BACKGROUND

Cancer patients who also have chronic inflammatory diseases, such asType 2 diabetes, have a higher risk of metastasis than patients with thesame stage and type of cancer who have normal immunometabolism,particularly in breast and prostate cancers. Yet cancer patientmetabolism, medications and adipocyte or bone health are typically notconsidered in evaluating risk for progression or metastasis of thesecancers. The >100 million Americans who are diabetic or pre-diabetic atpresent are insufficiently served by the standard of care in oncology.Clinical decision making could be greatly improved for patients at-riskfor cancer progression on account of their metabolic co-morbidities, butthis requires novel diagnostic tools and patient treatment paradigms.

SUMMARY

Most cancer biomarkers rely on markers derived from or induced by cancercells. Remarkably, the system described herein to assess cancer riskrelies on signals from non-tumor tissue which are shown to inducedangerous changes in cancer cells.

The inventors have found that small extracellular vesicles calledexosomes (about 30-90 nm in diameter) that originate in non-tumortissue, such as fat or bone, can carry molecular signals that promotemore dangerous (pro-metastatic) changes in cancer. These factors arealso found in the peripheral blood of cancer patients and can be used toprofile to assist clinical decision making about risks for progressionand metastasis.

In one aspect of any of the embodiments, described herein is a methodcomprising: determining the expression of at least one gene selectedfrom the group consisting of: miR374a-5p, miR-93-5p, miR-28-3p,miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug(SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, AHNAK, miR-424-5p,miR-326, miR424-5p, miR-27a-3p, miR320b, and miR320d; in an exosomeobtained from a subject.

In some embodiments of any of the aspects, the expression of at leastone gene selected from the group consisting of: miR374a-5p, miR-93-5p,miR-28-3p, miR-let-7b-3p, miR-375, miR-424-5p, miR-326, miR424-5p,miR-27a-3p, miR320b, and miR320d; is determined. In some embodiments ofany of the aspects, the expression of at least one gene selected fromthe group consisting of: miR374a-5p, miR-93-5p, miR-28-3p,miR-let-7b-3p, and miR-375; is determined. In some embodiments of any ofthe aspects, the expression of at least two genes selected from thegroup consisting of: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p,and miR-375; is determined. In some embodiments of any of the aspects,the expression of at least three genes selected from the groupconsisting of: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, andmiR-375; is determined. In some embodiments of any of the aspects, theexpression of at least four genes selected from the group consisting of:miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, and miR-375; isdetermined. In some embodiments of any of the aspects, the expression ofat least miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, and miR-375;is determined. In some embodiments of any of the aspects, the expressionof at least miR374a-5p is determined.

In some embodiments of any of the aspects, an increased level ofexpression of at least one gene selected from: miR374a-5p, miR-93-5p,miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist (TWIST1),Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, and AHNAK; and/ora decreased level of expression of at least one gene selected from:miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d;indicates an increased risk of cancer, metastasis, and/or EMT for thesubject, wherein the level of expression is relative to the level ofexpression in a exosome obtained from a healthy non-diabetic subject.

In some embodiments of any of the aspects, the method further comprisesa) i) administering a glucose-controlling medication or obesitymedication and/or ii) administering CT scans at a frequency of higherthan 1 CT scan every 6 months, to a subject determined to have anexpression level of at least one gene selected from: miR374a-5p,miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist(TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, andAHNAK which is increased relative to a reference; or an expression levelof at least one gene selected from: miR424-5p, miR-326, miR424-5p,miR-27a-3p, miR320b and miR320d; which is decreased relative to areference.

In some embodiments of any of the aspects, the method further comprisesa) i) administering a glucose-controlling medication or obesitymedication and/or ii) administering CT scans at a frequency of higherthan 1 CT scan every 6 months, to a subject determined to have anexpression level of at least one gene selected from: miR374a-5p,miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist(TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, andAHNAK which is increased relative to a reference; or an expression levelof at least one gene selected from: miR424-5p, miR-326, miR424-5p,miR-27a-3p, miR320b and miR320d; which is decreased relative to areference; or b) i) not administering a glucose-controlling medicationor obesity medication and/or ii) administering CT scans at a frequencyof no more than 1 CT scan every 6 months, to a subject determined tohave an expression level of at least one gene selected from: miR374a-5p,miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist(TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, andAHNAK which is not increased relative to a reference; or an expressionlevel of at least one gene selected from: miR424-5p, miR-326, miR424-5p,miR-27a-3p, miR320b and miR320d; which is not decreased relative to areference.

In one aspect of any of the embodiments, described herein is a method oftreating cancer, comprising: a) i) administering a glucose-controllingmedication or obesity medication and/or ii) administering CT scans at afrequency of higher than 1 CT scan every 6 months, to a subjectdetermined to have an expression level of at least one gene selectedfrom: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5,Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin(CDH1), ZEB1, and AHNAK which is increased relative to a reference; oran expression level of at least one gene selected from: miR424-5p,miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d; which is decreasedrelative to a reference.

In one aspect of any of the embodiments, described herien is a method oftreating cancer, comprising: a) i) administering a glucose-controllingmedication or obesity medication and/or ii) administering CT scans at afrequency of higher than 1 CT scan every 6 months, to a subjectdetermined to have an expression level of at least one gene selectedfrom: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5,Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin(CDH1), ZEB1, and AHNAK which is increased relative to a reference; oran expression level of at least one gene selected from: miR424-5p,miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d; which is decreasedrelative to a reference; orb) i) not administering a glucose-controllingmedication or obesity medication and/or ii) administering CT scans at afrequency of no more than 1 CT scan every 6 months, to a subjectdetermined to have an expression level of at least one gene selectedfrom: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5,Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin(CDH1), ZEB1, and AHNAK which is not increased relative to a reference;or an expression level of at least one gene selected from: miR424-5p,miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d; which is notdecreased relative to a reference.

In some embodiments of any of the aspects, the glucose-controllingmedication is selected from the group consisting of: metformin, asulfonylurea, a glinide, a SGLT2 inhibitor, and insulin. In someembodiments of any of the aspects, the glucose-controlling medication ismetformin. In some embodiments of any of the aspects, the obesitymedication selected from the group consisting of: orlistat,phentermine-topiramate, naltrexone-bupropion, liraglutide, semagludtide,setmelanotide, phentermine, benzphetamine, diethylpropion, andphendimetrazine.

In some embodiments of any of the aspects, the level of expression isthe level of mRNA. In some embodiments of any of the aspects, theexosome is 30-90 nm in diameter. In some embodiments of any of theaspects, the exosome originates from a non-tumor tissue. In someembodiments of any of the aspects, the exosome is isolated from anon-tumor tissue and/or cells. In some embodiments of any of theaspects, the non-tumor tissue and/or cells is blood, plasma, adiposetissue, adipocytes, or bone.

In some embodiments of any of the aspects, the method further comprisesdetermining the expression level of at least one gene selected fromCOMP, TSP5, BRD2, BRD3, miR103a, and SOX2-OT in tumor tissue obtainedfrom the subject.

In some embodiments of any of the aspects, the cancer is an epithelialcancer. In some embodiments of any of the aspects, the cancer is anepithelial adenocarcinoma. In some embodiments of any of the aspects,the cancer is esophageal cancer, pancreatic cancer, cervical cancer,colorectal cancer, gastric cancer, lung cancer, uterine caner, renalcancer, breast cancer, or prostate cancer. In some embodiments of any ofthe aspects, the cancer is breast and/or prostate cancer.

In some embodiments of any of the aspects, the subject is diabetic,overweight, and/or obese. In some embodiments of any of the aspects, thesubject is identified as diabetic when they are determined to have HbA1cof 6.5% or greater, or by fasting glucose or fasting insulin.

In one aspect of any of the embodiments, described herein is a method oftreating cancer in a subject in need thereof, the method comprisingadministering to the subject exosomes which are: from a non-diabeticand/or non-obese donor; and/or determined to have an level of expressionof at least one gene selected from: miR374a-5p, miR-93-5p, miR-28-3p,miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug(SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, and AHNAK; which isnot increased; and/or a level of expression of at least one geneselected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b andmiR320d; which is not increased, wherein the level of expression isrelative to the level of expression in a exosome obtained from a healthynon-diabetic subject.

In one aspect of any of the embodiments, described herein is a method oftreating cancer in a subject in need thereof, the method comprisingadministering to the subject: an inhibitor of at least one gene selectedfrom: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5,Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin(CDH1), ZEB1, and AHNAK; which is not increased; and/or an agonist of atleast one gene selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p,miR320b and miR320d.

In some embodiments of any of the aspects, the subject is one determinedto have: an increased level of expression of at least one gene selectedfrom:miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5,Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin(CDH1), ZEB1, and AHNAK; or a decreased level of expression of at leastone gene selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p,miR320b and miR320d.

In some embodiments of any of the aspects, the method results in EMTbeing reduced in the subject. In some embodiments of any of the aspects,the subject is further administered a BET inhibitor or PROTAC degrader.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D demonstrate that human adipocytes induce transcription ofEMT genes in co-cultured ER+ human breast cancer cell model. (FIG. 1A)MCF-7 cells were co-cultured for 5 days with insulin sensitive (IS)adipocytes that were differentiated from human primary pre-adipocytes,or ex vivo-induced insulin resistant (IR) adipocytes from the samesource, and compared to control without coculture (con). Expression ofselected EMT genes was analyzed by commercial PCR array (n=3). (FIG. 1B)Ingenuity pathway analysis of differentially expressed MCF-7 genes in(FIG. 1A) revealed that, compared to IS adipocytes, co-culture with IRadipocytes strongly induced tumor cell signatures associated withaggressiveness. (FIG. 1C) Fluorimetric glucose uptake assay confirmedthat 24 h treatment of primary adipocytes with recombinant human TNFα(250 pM; +) vs untreated control (−) converted IS to IR adipocytes,unable to transport glucose in response to 10 nM insulin. (FIG. 1D)Extracellular vesicles (EV) purified from adipocyte conditioned mediainduced transcription of EMT genes SNAI1 and SNAI2, but not EVs purifiedfrom conditioned media of 5 matched, undifferentiated pre-adipocytes(pre). Fold-change of selected genes Snail (SNAI1) and Slug (SNAI2) wasmeasured by RT-PCR relative to β-actin (ACTB) with qPCR TaqMan probes(n=3; *, P<0.05; ***, P<0.005; ns, not significant).

FIGS. 2A-2F demonstrate that EVs from adipocytes of T2D patients showgreater induction of EMT and cancer stem-like cell (CSC) genes in breastcancer models than EVs from adipocytes of ND patients. Human primarysubcutaneous pre-adipocytes from T2D and non-diabetic (ND) patients weredifferentiated and EVs were isolated from conditioned media. (FIG. 2A)MCF-7 cells were treated with EVs for 5 days and Ingenuity pathwayanalysis was performed on EMT gene expression array data (n=3). (FIG.2B) Compared to control or ND adipocytes, EVs from T2D adipocytesincreased expression of N-cadherin and decreased expression ofE-cadherin in MCF-7 cells, as analyzed by immunofluorescence with DAPIcounterstain. Bar, 10 μm (FIG. 2C) Similarly, EVs from T2D patientadipocytes upregulated pathways associated with cancer stem cellformation (n=3). (FIG. 2D) Individual EMT genes from (FIG. 2A) werevalidated with PCR TaqMan probes. (FIG. 2E) Individual CSC genes from(FIG. 2C) were validated with PCR TaqMan probes. (FIG. 2F) Quantitationof FIG. 2B, showing downregulation of E-cadherin and upregulation ofN-cadherin by T2D adipocyte EVs, compared to ND and controls. (n=3; *,P<0.05; **, P<0.01; ***, P<0.005; ns, not significant).

FIGS. 3A-3F demonstrate that EVs from adipocytes of T2D patients inducemorphological, transcriptional and migration phenotypes characteristicof EMT in human breast cancer cells, compared to EVs from adipocytes ofND patients. (FIG. 3A) Treatment for 5 days of three human breast cancercell lines (MCF-7, T47D, MDA-MB-231) with EVs from adipocytes of T2Dpatients compared to ND patients or to untreated control (con) alteredmorphology by light microscopy. Bar, 30 μm. (FIG. 3B) increased cellularperimeter compared to EVs from adipocytes of ND patients and untreatedcontrols, decreased cellular circularity, and increased cellularelongation, a parameter which is converse to circularity. Morphologicalanalysis was conducted using ImageJ. (FIG. 3C) EVs from T2D adipocytesincreased migration of MDA-MB-231 breast cancer cells in a 6-h transwellassay. Cells that reached the distal side of the 0.8-μm pore membranewere visualized by crystal violet stain and microscopy. (FIG. 3D)Quantitation of FIG. 3C. (FIG. 3E) Unbiased proteomic analysis of humanadipocyte EVs by LC-MS/MS. Σ# PSM (number of Peptide-Spectrum Matches)reports the sum of occurrences of unique peptides for each protein.(FIG. 3F) Knockdown of BRD2 and BRD4 ablated the ability of EVs fromeither T2D or ND adipocytes to upregulate SNAI and SNAI2 in MCF-7 cells,but not knockdown of BRD3. Gene expression is relative to (β-actin andcompared to scrambled siRNA control (scr) (n=3; *, P<0.05; **, P<0.01;***, P<0.001).

FIGS. 4A-4E demonstrate that TSP5 protein drives expression of EMTphenotypes. Pre-adipocytes were transduced with TSP5-expressinglentivirus and differentiated to mature adipocytes as above. (FIG. 4A)RT-PCR confirmed increased transcript and Elispot confirmed increasedprotein compared to EV marker CD63. (FIG. 4B) Upon addition to MCF-7cells, TSP5 EVs induced EMT genes ZEB1 and SNAI2 compared to control EVsfrom adipocytes that had been transduced with empty vector lentivirus.Induction of ZEB1 and SNAI2 by human recombinant transforming growthfactor (TGF)-β is shown as a positive control. (FIG. 4C) Similar to FIG.2, synthetic cationic vesicles loaded with ecombinant human TSP5 wereadded to MCF-7 cells for 5 days and EMT induction as measured byincreased vimentin expression was measured by IHC. Anti-Tsp5 antibodywas used to confirm delivery of protein by the synthetic vesicle system.As a control for loading and delivery, fluorescently labeled ovalbuminwas also incorporated into the vesicles. Bar, 10 μm (FIG. 4D) Heatmap ofgenes induced by TSP5-loaded vesicles confirms induction of EMT and MMPgenes. (FIG. 4E) Ingenuity analysis of TSP5 treatment confirmsupregulation of invasion and migration pathways and downregulation ofcell death pathways.

FIG. 5 depicts Kaplan-Meier curves of distant metastasis-free survivalof breast cancer patients, calculated from TCGA data. The level ofexpression of COMP, the gene encoding TSP5, was assessed for associationwith distant metastasis-free survival (DMFS) in breast cancer patientsin co-expression with each of the three somatic BET bromodomain genesBRD2, BRD3 or BRD4. Expression groups of equal size were characterizedas high (grey) or low (black) for each gene, and 10 joint probabilitieswere computed for 873 patients per group. Survival times are shown inmonths; hazard ratios (HR) and confidence intervals (in brackets) areshown for each pair of genes. A statistically significant, 35% increasedrisk of distant metastasis over 25 years was determined for highco-expression of BRD2 and COMP, and a 19% increased risk for highco-expression of BRD3 and COMP. There was a trend to DMFS for high BRD4and COMP, but the difference did not reach statistical significance for332 patients per group.

FIGS. 6A-6C depict the selectivity of EMT signatures induced by insulinresistant and insulin sensitive adipocytes. (FIG. 6A) MCF-7 cellstreated with EVs purified from adipocyte conditioned media inducetranscriptional upregulation of an individual mesenchymal marker gene(NOTCH1), decrease of an epithelial marker gene (CDH1) or no change ofcontrol genes (STAT3 and ERBB3) by RT-PCR of mRNA using qPCR TaqManprobes. (con, media control EVs; IS, insulin sensitive adipocyte EVs;IR, insulin resistant adipocyte EVs) (n=3; *, P<0.05; ***, P<0.005; ns,not significant) (FIG. 6B) Ingenuity pathway analysis of MCF-7 genesfrom Fig lA co-culture, comparing IS to IR adipocytes, arrangedaccording to Z-score for upregulation in IR. (FIG. 6C) NanoSight sizefractionation of adipocyte-origin exosomes. Total exosomes were purifiedfrom conditioned media of human primary adipocyte (ABM-007) cultures,resuspended in phosphate buffered saline pH 7.4 without divalent cationsand then analyzed by NanoSight to determine concentration and particlesizes. The major population of extracellular vesicles from insulinsensitive and insulin resistant adipocytes had similar diameter of 40-60nm.

FIGS. 7A-7C depict Ingenuity pathway analysis of MCF-7 genes induced byEVs from adipocytes of ND patients compared to T2D patients. (FIG. 7A)Full array results of EMT gene panel. (FIG. 7B) Full array results ofCSC gene panel. Genes that are shared between the two panels areidentified by arrows in matching color. Note the rank order of theshared genes is conserved between the two arrays. Heat maps shown fortriplicate samples. (FIG. 7C) NanoSight size characterization of primaryadipocyte-origin exosomes. Exosomes were purified from conditioned mediaof human primary adipocyte (ND, ABM-007) and (T2D, ABM-004) cultures,resuspended in PBS pH 7.4 without divalent cations and then analyzed byNanoSight to determine concentration and particle sizes. The majorpopulation of extracellular vesicles from ND and T2D adipocytes hadsimilar diameter of 50-150 nm.

FIG. 8 depicts Ingenuity Pathway Analysis of proteomics signatures.Peptide signals from proteomics analysis of human primary adipocyteexosomes were compared by analysis of signaling and functional pathways,with insulin sensitive (IS) and insulin resistant (IR) states as theindependent variables. Consistent with FIG. 1B, selected pathwaysassociated with cell migration and invasion were upregulated in the IRcondition compared to IS, whereas selected pathways associated withapoptosis were downregulated.

FIGS. 9A-9D depict EMT induction in murine 4T1 cells treated with EVsfrom IS vs IR murine adipocytes. Murine 3T3-L1 adipocytes weredifferentiated from pre-adipocyte fibroblasts as published (24), treatedwith 250 pM murine TNFα overnight, as specified to induce insulinresistance (9), then adipocyte conditioned media was used as the sourceof EVs. (FIG. 9A) 4T1 cells were treated for 3 days with EVs from IS orIR adipocytes, vs. media control EVs, then cells were fixed forimmunohistochemistry. Vimentin and E-cadherin, as mesenchymal orepithelial markers respectively, were visualized byfluorophore-conjugated primary anti-mouse antibodies, with phalloidinstain for cytoskeletal structure and DAPI counterstain for DNA. Bar, 10μm. (FIG. 9B) RT-PCR validation of two selected EMT genes, induced byEVs. (FIG. 9C) Transwell migration assay with 4T1 cells exposed to IS vsIR EVs, or IR treated with 50 nM MZ-1 (25) to inhibit BRD4 and blockmigration (18,24). (FIG. 9D) Quantitation of B. (n=3; ***, P<0.005).

FIGS. 10A-10B demonstrate that TSP5 overexpression in MDA-MD-231 cellsreduces expression of epithelial marker protein E-cadherin and increasesexpression of mesenchymal marker protein Vimentin. (FIG. 10A) V5-tagrecombinant TSP5 protein (COMP) was overexpressed in MDA-MB-231 cellsand IF was performed after 5 days to detect TSP5 (FIG. 10A), and changesin expression of E-cadherin (FIG. 10A) or Vimentin (FIG. 10B). Cellswere counterstained with phalloidin and DAPI. Quantitation of IFvisualization is shown by mean staining intensity (pixels/μm). Bar, 10μm. (n=3; *, P<0.05; **, P<0.01; ***, P<0.005)

FIGS. 11A-11C demonstrate that TSP5 (COMP) overexpression in MCF10Acells increased CSC markers. TSP5 overexpression induced a high ratio ofCD44hi/CD24 expression as determined by flow cytometry, consistent withenhanced invasion and metastatic potential. (n=3; ****, P<0.0001).

FIGS. 12A-12D demonstrate that TSP5 knockdown in 3T3-L1 adipocytesreduced the Tsp5 content of mature adipocyte exosomes, andcorrespondingly reduced exosome induction of selected EMT gene, Zeb1.Murine 3T3-L1 pre-adipocyte fibroblasts were exposed to shRNA againstmurine Tsp5, then differentiated into mature adipocytes as above.Conditioned media of these cells was used as the sources of exosomes forexperiments with 4T1 cells. (FIG. 12A) Tsp5 mRNA from Tsp5 knockdownadipocytes is compared by qPCR to control adipocytes. (FIG. 12B) Elispotof exosomes purified from scrambled control adipocytes compared toelispot of exosomes purified from Tsp5 knockdown adipocytes, with Cd63as an exosome marker vs. Tsp5. (FIG. 12C) Quantitation of FIG. 12B.(FIG. 12D) Negative control experiment shows that if exosomes (EV) aredepleted of Tsp5 through knockdown of the gene in the source adipocytes,Zeb1 is no longer induced in 4T1 cells. Comparison is to exosomespurified from control knockdown adipocytes (scr), and non- conditionedmedia exosomes as reference (media). TGF-β is shown as a positivecontrol for induction of Zeb1. A parallel experiment with commerciallyavailable, conditioned media that had been depleted of exosomes before4T1 cell culture caused cell stress in the assay (results not shown),thus this type of experiment was not suitable as an additional negativecontrol. (n=3; *, P<0.05; **, P<0.01) (scr, scrambled shRNA control;TGF-β, transforming growth factor-β).

FIG. 13 depicts a table of clinical information of donors of primary,subcutaneous pre-adipocytes.

FIG. 14 depicts a table of Proteomics analysis of total exosomespurified from adipocyte-conditioned media. Insulin sensitive adipocytesare from a non-diabetic person, converted to insulin resistant ex vivoafter treatment with TNF-α. Proteins are ranked based on the PSMdifference between ND and T2D. ND, non-diabetic; T2D, Type 2 diabetic;IS, insulin sensitive; IR, insulin resistant; PSMs (number ofPeptide-Spectrum Matches), total number of occurrences of uniquepeptides for each protein; AAs, the number of amino acids in theprotein; MW, molecular weight of the protein in kDa; Calc. pI,isoelectric point of each protein.

FIGS. 15A-15C demonstrate that human plasma exosomes from T2D subjectsinduce EMT genes in prostate cancer cell lines. The DU145 human prostatecancer cell line was treated with either ND or T2D plasma exosomes (109)for 2 days. (FIG. 15A) Plasma exosomes from four, independent T2Dsubjects (▴) increased SNAI1 mRNA expression after normalizing to ACTBof the respective sample and compared to the control cells that weretreated with growth media exosomes. (●). Exosomes from four, independentND subjects (▪) did not increase SNAI1 mRNA expression relative to ACTBcontrol. TGF-β (TGFB, 5 ng/mL) was used as a positive control (▾) forinduction of pro-EMT gene transcription or repression of pro-MET genetranscription. (FIG. 15B) Plasma exosomes from T2D subjects did notinduce CDH1 expression. ND plasma exosomes increased CDH1 mRNAexpression. Data in FIGS. 15A and 15B were obtained from four biologicalreplicates of ND and T2D, and each biological replicate was conducted inthree technical replicates, that are averaged in the graph. Data wereanalyzed by two-way ANOVA with statistical significance presented as: *,P=0.0244; and ****, P<0.0001; ns, not significant. (FIG. 15C) Expressionof selected EMT genes were analyzed by commercial PCR array. Relativeexpression of significantly differentially expressed genes in threeindependent, T2D exosome-treated samples was compared to threeindependent, ND exosome-treated samples. Equal numbers of exosomes (10⁹)from each sample were used. The heatmap of the PCR array result wascalculated by hierarchical clustering. Scale bar (right) shows foldchange. (ND, non-diabetic; T2D, Type 2 diabetic; TGFB, TGF-β positivecontrol).

FIGS. 16A-16C demonstrate that plasma exosomes from T2D subjects inducemajor features of tumor cell aggressive behavior. (FIG. 16A) Ingenuitypathway analysis (IPA) of disease and function based on FIG. 15C. IPAprediction shows that plasma exosomes from independent T2D subjectsstrongly induced tumor cell signatures associated with canceraggressiveness compared to plasma exosomes from independent ND subjects.(FIG. 16B) Morphology of DU145 cells treated with equal number of plasmaexosomes ND and T2D (10⁹) compared to the control cells treated withgrowth media (RPMI-1640+10% FBS) exosomes. One representative field ofview is shown, out of 25 images collected for each of the threeexperimental conditions with three replicates. Scale bar, 30 μm. (FIG.16C) Quantification of cell morphology, including cellular perimeter,circularity and elongation (a parameter that is converse to circularity)measured in images from FIG. 16B. Expression in each experimental wascompared to control (n=25 cells each from N=3 independent experiments).Data were analyzed by one-way ANOVA, with statistical significancepresented as: ****, p<0.0001. (ND, non-diabetic; T2D, Type 2 diabetic;Control, media-only exosomes).

FIGS. 17A-17C demonstrate that T2D plasma exosomes upregulate genes thatencode immune checkpoint ligands in prostate cancer cells. Plasmaexosomes from T2D subjects (▴) upregulated CD274 (FIG. 17A) and CD155(FIG. 17B) mRNA expression in DU145 cells after 2 days, compared to NDcontrol (▪). Treatment with IFN-γ (5 ng/mL) was the positive control forCD274 induction (▾), as previously published [53]. Treatment with growthmedia exosomes was the negative control (●). Data in FIG. 17A and 17Bwere obtained from two biological replicates of ND and T2D, and eachbiological replicate was conducted in three technical replicates. Datawere analyzed by two-way ANOVA with statistical significance presentedas: *, P<0.05; ns, not significant. (FIG. 17C) Hierarchical clusteringof genes involved in inflammation and cancer immune crosstalk, analyzedby commercial PCR array. DU145 cells were treated with plasma exosomesfrom three T2D subjects or three ND subjects. Equal numbers of exosomes(10⁹) from each sample were used. The heatmap of the PCR array resultwas calculated by hierarchical clustering. Scale bar (right) shows foldchange. (ND, non-diabetic; T2D, Type 2 diabetic; Control, media-onlyexosomes; IFNgamma, interferon-γ).

FIGS. 18A-18D demonstrate that T2D plasma exosomes have distinctmicroRNA profile compared to the ND plasma exosomes. (FIG. 18A) Heatmapof differentially expressed microRNAs in plasma exosomes of two T2Dsubjects compared to plasma exosomes of ND subjects. Total RNA wasisolated from plasma exosomes of T2D and ND subjects, and miRNAs wereprofiled by a commercial PCR array. DU145 cells were transfected withindividual miRNAs from (FIG. 18A) (25 nM), selected based on theirfunctional significance for EMT and immune exhaustion. The mRNAexpression of SNAI1 (FIG. 18B), CD274 (FIG. 18C) and CDH1 (FIG. 18D) wastested and expression relative to β-actin (ACTB) is shown. Scale bar(FIG. 18A, right) shows fold change. Data were analyzed by one-way ANOVAwith statistical significance presented as: *, P<0.05; ***, P<0.001;****, P<0.0001; ns, not significant. (Control, media only exosomes;TGFB, TGF-β positive control at 5 ng/mL for SNAI1 induction; IFNgamma,interferon-γ positive control at 5 ng/mL for CD274 induction).

FIGS. 19A-19C demonstrate that T2D plasma exosomes require BRD4 toupregulate EMT genes and PD-L1 expression. (FIGS. 19A) Expression ofSNAI1 and CD274 genes in DU145 cells measured by RT-PCR of cellular RNAafter exosome treatment. T2D exosomes (109 ; T2D Exo) were compared tomedia only control exosomes (Control), or T2D Exo+JQ1 or MZ1. (JQ1 is apan-BET inhibitor (400 nM) and MZ-1 is a BRD4-selective degrader (50nM)). (FIG. 19B) Expression of PD-L1 was measured by flow cytometryafter treatments. One million events were analyzed by Flow-Jo andpresented as overlaid histograms (control, red trace; experimental,black trace). (FIG. 19C) Flow cytometry data of FIG. 19B were quantifiedwith PD-L1 as percent of parent population. The experiment was conductedin triplicate with differences between means represented as bar graphs.Data were analyzed by two-way ANOVA with statistical significancepresented as: ****, P<0.0001 (TGFB, TGF-β positive control at 5 ng/mLfor SNAI1 induction; IFNgamma, interferon-γ positive control at 5 ng/mLfor CD274 induction).

FIG. 20 depicts a model for T2D exosome delivery of miRNAs thatreprogram transcription of tumor cell genes important for canceraggressiveness. Circulating exosomes in plasma arrive at the tumor cellsurface where they are internalized and release their cargo of miRNAs.These miRNAs are trafficked to the nucleus where they reprogram signaltransduction pathways. In this case, miR-93-5p and miR374a-5p are shownsignaling through BRD4, which is an essential transcriptionalco-regulator of genes important for tumor cell aggressiveness, SNAI1,CDH1 and CD274.

FIGS. 21A-21B depict T2D exosome induction of selected EMT genes,determined by RTPCR.Human plasma exosomes from T2D subjects (▴) inducedrepresentative EMT genes ANHAK (FIG. 21A) and VIMENTIN (Vim) (FIG. 21B)in DU145 cells, shown as fold-change relative to ACTB housekeeping gene(●). Responses for plasma exosomes from T2D subjects are compared to NDcontrols (▪). Data were obtained from two biological replicates of NDand T2D; each biological replicate was conducted in three technicalreplicates. Data were analyzed by twoway ANOVA, with statisticalsignificance presented as: **, p<0.01; ***, p<0.001; ****,p<0.0001; ns,not significant. ND, non-diabetic; T2D, Type 2 diabetic; TGFB, TGF-βpositive control (▾).

FIG. 22 depicts Ingenuity pathway analysis (IPA) of disease andfunction. IPA prediction based on FIG. 17C shows that plasma exosomesfrom T2D subjects induced major mechanisms associated with angiogenesis,immune dysfunction and tumor progression, compared to plasma exosomesfrom ND subjects. Three independent datasets for T2D are compared tothree independent datasets for ND. Scale bar (right) shows fold change.(ND, non-diabetic; T2D, Type 2 diabetic)

FIGS. 23A-23B depict the time course of exosomal RNA uptake into DU145cell nuclei. (FIG. 23A) Exosome RNA was stained with RNASelect (Exo RNA;Alexa Fluor™-488), then exosomes were washed and added to DU145 cells.Cells were imaged at 0, 4, 8, 16 and 24-hour time points. Filamentousactin (F-actin) was stained with Alexa Fluor™ Phalloidin probe 568 nm(Phalloidin) and DNA was visualized by DAPI counterstain (DAPI). Onerepresentative field of view is shown, out of 25 images collected foreach of the three experimental conditions with three replicates. Scalebar, 10 μm. The mean intensity of stained RNA (Alexa Fluor™-488) wasquantified using ImageJ (FIG. 23B).

FIGS. 24A-24C depict genome-wide transcriptional analysis by RNA seq.(FIG. 24A) Plot of PC2 vs. PC1, computed across all genes in the DU145samples. Plasma exosomes of T2D subjects had unique and different PCAvalues, while plasma exosomes of ND subjects had PCA values that wereclose to the media-only control values. (FIG. 24B) RNA seq expression ofmiR103a, SOX2-OT, Cav1 and SNAI1 in DU145 cells treated with ND and T2Dexosomes. The variance stabilizing transformed (VST) expression valuesfor each gene were z-score-normalized to a mean of zero and standarddeviation of 1 within all replicates of all samples. The Y axis showsthe VST values. (FIG. 24C) Differential expression of genes unregulatedin DU145 cells treated with T2D exosomes (right) vs. those of NDexosomes (left). Significantly differentially expressed genes wereidentified using a p value cutoff of 0.05 and a fold change cutoff of 1(dotted lines). Figure was generated using EnhancedVolcano package.

DETAILED DESCRIPTION

As described herein, the inventors have found that exosomes withspecific gene expression patterns influence cancer cell behavior,promoting EMT and metastasis. The inventors demonstrate that this geneexpression profile can be used as a biomarker to detect patients withcancer, high risk cancer, or who are at high risk of cancer, EMT, and/ormetastasis. The inventors also describe methods of treatment, includingadministering exosomes without the pro-metastasis gene expressionprofile, or by administering treatments that counteract the crosstalk ofthe cancer promoting exosomes and cancer cells.

Accordingly, in one aspect of any of the embodiments, described hereinis a method comprising determining the expression of at least one geneselected from the group consisting of: miR374a-5p, miR-93-5p, miR-28-3p,miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug(SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, AHNAK, miR-424-5p,miR-326, miR424-5p, miR-27a-3p, miR320b, and miR320d; in an exosomeobtained from a subject.

As used herein, “exosome” refers to a nano- or micro-sized membranevesicle vesicle comprising a membrane that encloses an internal space,and which is secreted or shed from a cell, e.g., by direct plasmamembrane budding or by fusion of the late endosome with the plasmamembrane. Exosomes are enclosed by a lipid bilayer and range in sizefrom approximately 10 nm to 1000 nm in diameter. Without wishing to bebound by theory, it is believed that materials endocytosed by a cell orother cellular components may be sorted into endosomal compartments,forming intraluminal vesicles (multivesicular endosomes ormultivesicular bodies (MVBs)). The vesicles may then be released intothe extracellular environment upon the fusion of MVBs with the plasmamembrane.

In some embodiments of any of the aspects, the exosome is from 5-200 nmin diameter. In some embodiments of any of the aspects, the exosome isfrom 10-150 nm in diameter. In some embodiments of any of the aspects,the exosome is from 20-110 nm in diameter. In some embodiments of any ofthe aspects, the exosome is from 30-90 nm in diameter.

In some embodiments of any of the aspects, the method further comprisesa first step of size selection or purification of exosomes from a sampleobtained from the subject, e.g., to provide a sample comprising onlyexosomes of 5-200 nm diameter size, 10-150 nm diameter size, 20-110 nmdiameter size, or 30-90 nm diameter size. In some emobdiments of any ofthe aspects, the sample does not comprise cells. In some emobdiments ofany of the aspects, the sample does not comprise cell-free DNA.

In some embodiments of any of the aspects, the exosome originated from anon-tumor tissue and/or cells. In some embodiments of any of theaspects, the exosome is isolated, purified, or size-selected from anon-tumor tissue and/or cells. In some embodiments of any of theaspects, the exosome is isolated, purified, or size-selected from asample comprising non-tumor tissue and/or cells. In some embodiments ofany of the aspects, the exosome is isolated, purified, or size-selectedfrom a sample taken from a non-tumor tissue. In some embodiments of anyof the aspects, the non-tumor tissue and/or cells is blood, plasma,adipose tissue, adipocytes, or bone. In some embodiments of any of theaspects, sample is or comprises blood, plasma, adipose tissue,adipocytes, or bone. In some embodiments of any of the aspects, thenon-tumor tissue and/or cells is blood or plasma. In some embodiments ofany of the aspects, sample is or comprises blood or plasma. In someembodiments of any of the aspects, the non-tumor tissue and/or cells isplasma. In some embodiments of any of the aspects, sample is orcomprises plasma.

In some embodiments of any of the aspects, the method comprisesdetermining the expression of at least two genes selected from the groupconsisting of: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),E-cadherin (CDH1), ZEB1, AHNAK, miR-424-5p, miR-326, miR424-5p,miR-27a-3p, miR320b, and miR320d. In some embodiments of any of theaspects, the method comprises determining the expression of at leastthree genes selected from the group consisting of: miR374a-5p,miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist(TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, AHNAK,miR-424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b, and miR320d. Insome embodiments of any of the aspects, the method comprises determiningthe expression of at least four genes selected from the group consistingof: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5,Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin(CDH1), ZEB1, AHNAK, miR-424-5p, miR-326, miR424-5p, miR-27a-3p,miR320b, and miR320d. In some embodiments of any of the aspects, themethod comprises determining the expression of at least five genesselected from the group consisting of: miR374a-5p, miR-93-5p, miR-28-3p,miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug(SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, AHNAK, miR-424-5p,miR-326, miR424-5p, miR-27a-3p, miR320b, and miR320d. In someembodiments of any of the aspects, the method comprises determining theexpression of at least five genes selected from the group consisting of:miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail(SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin(CDH1), ZEB1, AHNAK, miR-424-5p, miR-326, miR424-5p, miR-27a-3p,miR320b, and miR320d.

In some embodiments of any of the aspects, the method comprisesdetermining the expression of at least one gene selected from the groupconsisting of: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,miR-424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b, and miR320d; isdetermined. In some embodiments of any of the aspects, the methodcomprises determining the expression of at least two genes selected fromthe group consisting of: miR374a-5p, miR-93-5p, miR-28-3p,miR-let-7b-3p, miR-375, miR-424-5p, miR-326, miR424-5p, miR-27a-3p,miR320b, and miR320d; is determined. In some embodiments of any of theaspects, the method comprises determining the expression of at leastthree genes selected from the group consisting of: miR374a-5p,miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, miR-424-5p, miR-326,miR424-5p, miR-27a-3p, miR320b, and miR320d; is determined. In someembodiments of any of the aspects, the method comprises determining theexpression of at least four genes selected from the group consisting of:miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, miR-424-5p,miR-326, miR424-5p, miR-27a-3p, miR320b, and miR320d; is determined. Insome embodiments of any of the aspects, the method comprises determiningthe expression of at least five genes selected from the group consistingof: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,miR-424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b, and miR320d; isdetermined. In some embodiments of any of the aspects, the methodcomprises determining the expression of at least six genes selected fromthe group consisting of: miR374a-5p, miR-93-5p, miR-28-3p,miR-let-7b-3p, miR-375, miR-424-5p, miR-326, miR424-5p, miR-27a-3p,miR320b, and miR320d; is determined. In some embodiments of any of theaspects, the method comprises determining the expression of at leastseven genes selected from the group consisting of: miR374a-5p,miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, miR-424-5p, miR-326,miR424-5p, miR-27a-3p, miR320b, and miR320d; is determined. In someembodiments of any of the aspects, the method comprises determining theexpression of at least eight genes selected from the group consistingof: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,miR-424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b, and miR320d; isdetermined. In some embodiments of any of the aspects, the methodcomprises determining the expression of at least nine genes selectedfrom the group consisting of: miR374a-5p, miR-93-5p, miR-28-3p,miR-let-7b-3p, miR-375, miR-424-5p, miR-326, miR424-5p, miR-27a-3p,miR320b, and miR320d; is determined. In some embodiments of any of theaspects, the method comprises determining the expression of at least tengenes selected from the group consisting of: miR374a-5p, miR-93-5p,miR-28-3p, miR-let-7b-3p, miR-375, miR-424-5p, miR-326, miR424-5p,miR-27a-3p, miR320b, and miR320d; is determined. In some embodiments ofany of the aspects, the method comprises determining the expression ofat least miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,miR-424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b, and miR320d; isdetermined.

In some embodiments of any of the aspects, the method comprisesdetermining the expression of at least one gene selected from the groupconsisting of: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, andmiR-375; is determined. In some embodiments of any of the aspects, themethod comprises determining the expression of at least two genesselected from the group consisting of: miR374a-5p, miR-93-5p, miR-28-3p,miR-let-7b-3p, and miR-375; is determined. In some embodiments of any ofthe aspects, the method comprises determining the expression of at leastthree genes selected from the group consisting of: miR374a-5p,miR-93-5p, miR-28-3p, miR-let-7b-3p, and miR-375; is determined. In someembodiments of any of the aspects, the method comprises determining theexpression of at least four genes selected from the group consisting of:miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, and miR-375; isdetermined. In some embodiments of any of the aspects, the methodcomprises determining the expression of at least miR374a-5p, miR-93-5p,miR-28-3p, miR-let-7b-3p, and miR-375; is determined.

For example, in some embodiments of any of the aspects, the methodcomprises determining the expression of any of the followingcombinations of genes:

-   -   a) miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, and miR-375;    -   b) miR-93-5p, miR-28-3p, miR-let-7b-3p, and miR-375;    -   c) miR374a-5p, miR-28-3p, miR-let-7b-3p, and miR-375;    -   d) miR374a-5p, miR-93-5p, miR-let-7b-3p, and miR-375;    -   e) miR374a-5p, miR-93-5p, miR-28-3p, and miR-375;    -   f) miR374a-5p, miR-93-5p, miR-28-3p, and miR-let-7b-3p;    -   g) miR-28-3p, miR-let-7b-3p, and miR-375;    -   h) miR-93-5p, miR-let-7b-3p, and miR-375;    -   i) miR-93-5p, miR-28-3p, and miR-375;    -   j) miR-93-5p, miR-28-3p, and miR-let-7b-3p;    -   k) miR374a-5p, miR-let-7b-3p, and miR-375;    -   l) miR374a-5p, miR-28-3p, and miR-375;    -   m) miR374a-5p, miR-28-3p, and miR-let-7b-3p;    -   n) miR374a-5p, miR-93-5p, and miR-375;    -   o) miR374a-5p, miR-93-5p, and miR-let-7b-3p;    -   p) miR374a-5p, miR-93-5p, and miR-28-3p;    -   q) miR374a-5p, and miR-93-5p;    -   r) miR374a-5p, and miR-28-3p;    -   s) miR374a-5p, and miR-let-7b-3p;    -   t) miR374a-5p, and miR-375;    -   u) miR-93-5p and miR-28-3p;    -   v) miR-93-5p and miR-let-7b-3p;    -   w) miR-93-5p and miR-375;    -   x) miR-28-3p and miR-let-7b-3p;    -   y) miR-28-3p and miR-375; or    -   z) miR-let-7b-3p, and miR-375.

In some embodiments of any of the aspects, the method comprisesdetermining the expression of at least miR374a-5p. In some embodimentsof any of the aspects, the method comprises determining the expressionof at least miR374a-5p and at least one of miR-93-5p, miR-28-3p,miR-let-7b-3p, and miR-375.

The sequences for miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p,miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin(VIM), E-cadherin (CDH1), ZEB1, and AHNAK, miR424-5p, miR-326,miR424-5p, miR-27a-3p, miR320b and miR320d are known in the art. Forexample, the human sequences for the foregoing genes are available inthe NCBI and/or miRBase databases. Exemplary sequences for the foregoinggenes are provided in Table 3 below. In some embodiments of any of theaspects, miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5,Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin(CDH1), ZEB1, and AHNAK, miR424-5p, miR-326, miR424-5p, miR-27a-3p,miR320b or miR320d are a gene or gene expression product having asequence with at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or greater sequence identity to a sequence provided in Table3. In some embodiments of any of the aspects, miR374a-5p, miR-93-5p,miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist (TWIST1),Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, and AHNAK,miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b or miR320d are a geneor gene expression product having a sequence with at least 80%, at least85%, at least 90%, at least 95%, at least 98%, or greater sequenceidentity to a sequence provided in Table 3 and retaining the sameactivity as the reference sequence provided in Table 3. In someembodiments of any of the aspects, miR374a-5p, miR-93-5p, miR-28-3p,miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug(SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, and AHNAK, miR424-5p,miR-326, miR424-5p, miR-27a-3p, miR320b or miR320d are a gene or geneexpression product having the sequence of a sequence provided in Table3.

TABLE 3 Gene Human gene designation Exemplary Sequences As usedand/or full Accession SEQ ID  herein name Type Sequence or NCBI Gene IDNumber NO: miR374a- hsa-mir-374a RNA MI0000782 1 5p miR-93- hsa-miR-93-RNA MIMAT0000093 2 5p 5p miR-28- hsa-miR-28- RNA MIMAT0004502 3 3p 3pmiR-let- hsa-let-7b RNA MI0000063 4 7b-3p miR-375 hsa-mir-375 RNAMI0000783 5 miR424- hsa-miR-424- RNA MIMAT0001341 6 5p 5p miR-326hsa-mir-326 RNA MI0000808 7 miR-27a- hsa-miR-27a- RNA MIMAT0000084 8 3p3p miR320b hsa-miR-320b RNA This has a mature sequence MIMAT0005792 9 ofAAAAGCUGGGUUGAGA GGGCAA (SEQ ID NO: 9) and is encoded by the premiR-320b-1 and miR-320b- 2. miR320c hsa-miR-320c RNAThis has a mature sequence MIMAT0005793 10 of AAAAGCUGGGUUGAGAGGGU (SEQ ID NO: 10) and is encoded by the pre mir-320c-1 and mir-320c-2miR320d hsa-miR-320d RNA This has a mature sequence MIMAT0006764 11 ofAAAAGCUGGGUUGAGA GGA(SEQ ID NO: 11) and is encoded by the pre miR-320d-1 and miR-320d-2. TSP5 COMP mRNA ID: 1311 NM_000095.3 12 cartilageoligomeric matrix protein TSP5 COMP protein ID: 1311 NP_000086.2 13cartilage oligomeric matrix protein SNAI1 snail family mRNA ID: 6615NM_005985.4 14 transcriptional repressor 1 SNAI1 snail family proteinID: 6615 NP_005976.2 15 transcriptional repressor 1 TWIST1 twist familyRNA ID: 7291 NR_149001.2 16 bHLH transcription factor 1 TWIST1twist family mRNA ID: 7291 NM_000474.4 17 bHLH transcription factor 1TWIST1 twist family protein ID: 7291 NP_000465.1 18 bHLH transcriptionfactor 1 SNAI2 snail family mRNA ID: 6591 NM_003068.5 19 transcriptionalrepressor 2 SNAI2 snail family protein ID: 6591 NP_003059.1 20transcriptional repressor 2 VIM vimentin mRNA ID: 7431 NM_003380.5 21VIM vimentin protein ID: 7431 NP_003371.2 22 VIM Vimentin mRNA ID: 7431XM_006717500.2 23 isoform X1 VIM Vimentin protein ID: 7431XP_006717563.1 24 isoform X1 CDH1 cadherin 1 mRNA ID: 999 NM_001317184.225 CDH1 cadherin 1 Protein ID: 999 NP_001304113.1 26 CDH1 cadherin 1mRNA ID: 999 NM_001317185.2 27 CDH1 cadherin 1 Protein ID: 999NP_001304114.1 28 CDH1 cadherin 1 mRNA ID: 999 NM_001317186.2 29 CDH1cadherin 1 Protein ID: 999 NP_001304115.1 30 CDH1 cadherin 1 mRNAID: 999 NM_004360.5 31 CDH1 cadherin 1 protein ID: 999 NP_004351.1 32ZEB1, zinc finger E- mRNA ID: 6935 NM_001128128.3 33 box-bindinghomeobox 1 isoform a ZEB1 zinc finger E- Protein ID:6935 NP_001121600.134 box-binding homeobox 1 isoform c ZEB1 zinc finger E- mRNA ID: 6935NM_001174093.2 35 box-binding homeobox 1 isoform c ZEB1 zinc finger E-Protein ID: 6935 NP_001167564.1 36 box-binding homeobox 1 isoform d ZEB1zinc finger E- mRNA ID: 6935 NM_001174094.2 37 box-binding homeobox 1isoform d ZEB1 zinc finger E- Protein ID: 6935 NP_001167565.1 38box-binding homeobox 1 isoform e ZEB1 zinc finger E- mRNA ID: 6935NM_001174095.2 39 box-binding homeobox 1 isoform e ZEB1 zinc finger E-Protein ID: 6935 NP_001167566.1 40 box-binding homeobox 1 isoform f ZEB1zinc finger E- mRNA ID: 6935 NM_001174096.2 41 box-binding homeobox 1isoform f ZEB1 zinc finger E- Protein ID: 6935 NP_001167567.1 42box-binding homeobox 1 isoform g ZEB1 zinc finger E- mRNA ID: 6935NM_001323638.2 43 box-binding homeobox 1 isoform g ZEB1 zinc finger E-Protein ID: 6935 NP_001310567.1 44 box-binding homeobox 1 isoform g ZEB1zinc finger E- mRNA ID: 6935 NM_001323641.2 45 box-binding homeobox 1isoform g ZEB1 zinc finger E- Protein ID:6935 NP_001310570.1 46box-binding homeobox 1 isoform g ZEB1 zinc finger E- mRNA ID: 6935NM_001323642.2 47 box-binding homeobox 1 isoform g ZEB1 zinc finger E-Protein ID: 6935 NP_001310571.1 48 box-binding homeobox 1 isoform gZEB1m zinc finger E- mRNA ID: 6935 NM_001323643.2 49 box-bindinghomeobox 1 isoform g ZEB1 zinc finger E- Protein ID: 6935 NP_001310572.150 box-binding homeobox 1 isoform g ZEB1 zinc finger E- mRNA ID: 6935NM_001323644.2 51 box-binding homeobox 1 isoform g ZEB1 zinc finger E-Protein ID: 6935 NP_001310573.1 52 box-binding homeobox 1 isoform g ZEB1zinc finger E- mRNA ID: 6935 NM_001323645.2 53 box-binding homeobox 1isoform g ZEB1 zinc finger E- Protein ID: 6935 NP_001310574.1 54box-binding homeobox 1 isoform g ZEB1 zinc finger E- mRNA ID: 6935NM_001323646.2 55 box-binding homeobox 1 isoform g ZEB1 zinc finger E-Protein ID: 6935 NP_001310575.1 56 box-binding homeobox 1 isoform g ZEB1zinc finger E- mRNA ID: 6935 NM_001323647.2 57 box-binding homeobox 1isoform g ZEB1 zinc finger E- Protein ID: 6935 NP_001310576.1 58box-binding homeobox 1 isoform g ZEB1 zinc finger E- mRNA ID: 6935NM_001323648.2 59 box-binding homeobox 1 isoform g ZEB1 zinc finger E-Protein ID: 6935 NP_001310577.1 60 box-binding homeobox 1 isoform g ZEB1zinc finger E- mRNA ID: 6935 NM_001323649.2 61 box-binding homeobox 1isoform g ZEB1 zinc finger E- protein ID: 6935 NP_001310578.1 62box-binding homeobox 1 isoform g ZEB1 zinc finger E- mRNA ID: 6935NM_001323650.2 63 box-binding homeobox 1 isoform g ZEB1 zinc finger E-Protein ID: 6935 NP_001310579.1 64 box-binding homeobox 1 isoform g ZEB1zinc finger E- mRNA ID: 6935 NM_001323651.2 65 box-binding homeobox 1isoform g ZEB1 zinc finger E- protein ID: 6935 NP_001310580.1 66box-binding homeobox 1 isoform g ZEB1 zinc finger E- mRNA ID: 6935NM_001323652.2 67 box-binding homeobox 1 isoform g ZEB1 zinc finger E-protein ID: 6935 NP_001310581.1 68 box-binding homeobox 1 isoform g ZEB1zinc finger E- mRNA ID: 6935 NM_001323653.2 69 box-binding homeobox 1isoform g ZEB1 zinc finger E- protein ID: 6935 NP_001310582.1 70box-binding homeobox 1 isoform g ZEB1 zinc finger E- mRNA ID: 6935NM_001323654.2 71 box-binding homeobox 1 isoform g ZEB1 zinc finger E-Protein ID: 6935 NP_001310583.1 72 box-binding homeobox 1 isoform g ZEB1zinc finger E- mRNA ID: 6935 NM_001323655.2 73 box-binding homeobox 1isoform g ZEB1 zinc finger E- protein ID: 6935 NP_001310584.1 74box-binding homeobox 1 isoform g ZEB1 zinc finger E- mRNA ID: 6935NM_001323656.2 75 box-binding homeobox 1 isoform g ZEB1 zinc finger E-Protein ID: 6935 NP_001310585.1 76 box-binding homeobox 1 isoform g ZEB1zinc finger E- mRNA ID: 6935 NM_001323657.2 77 box-binding homeobox 1isoform g ZEB1 zinc finger E- Protein ID: 6935 NP_001310586.1 78box-binding homeobox 1 isoform g ZEB1 zinc finger E- mRNA ID: 6935NM_001323658.2 79 box-binding homeobox 1 isoform g ZEB1 zinc finger E-protein ID: 6935 NP_001310587.1 80 box-binding homeobox 1 isoform g ZEB1zinc finger E- mRNA ID: 6935 NM_001323659.2 81 box-binding homeobox 1isoform g ZEB1 zinc finger E- Protein ID: 6935 NP_001310588.1 82box-binding homeobox 1 isoform g ZEB1 zinc finger E- mRNA ID: 6935NM_001323660.2 83 box-binding homeobox 1 isoform g ZEB1 zinc finger E-protein ID: 6935 NP_001310589.1 84 box-binding homeobox 1 isoform g ZEB1zinc finger E- mRNA ID: 6935 NM_001323661.2 85 box-binding homeobox 1isoform g ZEB1 zinc finger E- protein ID: 6935 NP_001310590.1 86box-binding homeobox 1 isoform g ZEB1 zinc finger E- mRNA ID: 6935NM_001323662.2 87 box-binding homeobox 1 isoform g ZEB1 zinc finger E-protein ID: 6935 NP_001310591.1 88 box-binding homeobox 1 isoform g ZEB1zinc finger E- mRNA ID: 6935 NM_001323663.2 89 box-binding homeobox 1isoform g ZEB1 zinc finger E- protein ID: 6935 NP_001310592.1 90box-binding homeobox 1 isoform g ZEB1 zinc finger E- mRNA ID: 6935NM_001323664.2 91 box-binding homeobox 1 isoform g ZEB1 zinc finger E-protein ID: 6935 NP_001310593.1 92 box-binding homeobox 1 isoform g ZEB1zinc finger E- mRNA ID: 6935 NM_001323665.2 93 box-binding homeobox 1isoform g ZEB1 zinc finger E- protein ID: 6935 ID: 6935 NP_001310594.194 box-binding homeobox 1 isoform g ZEB1 zinc finger E- mRNA ID: 6935NM_001323666.2 95 box-binding homeobox 1 isoform g ZEB1 zinc finger E-protein ID: 6935 NP_001310595.1 96 box-binding homeobox 1 isoform g ZEB1zinc finger E- mRNA ID: 6935 NM_001323671.2 97 box-binding homeobox 1isoform g ZEB1 zinc finger E- protein ID: 6935 NP_001310600.1 98box-binding homeobox 1 isoform g ZEB1 zinc finger E- mRNA ID: 6935NM_001323672.2 99 box-binding homeobox 1 isoform g ZEB1 zinc finger E-protein ID: 6935 NP_001310601.1 100 box-binding homeobox 1 isoform gZEB1 zinc finger E- mRNA ID: 6935 NM_001323673.2 101 box-bindinghomeobox 1 isoform g ZEB1 zinc finger E- protein ID: 6935 NP_001310602.1102 box-binding homeobox 1 isoform g ZEB1 zinc finger E- mRNA ID: 6935NM_001323674.2 103 box-binding homeobox 1 isoform h ZEB1 zinc finger E-protein ID: 6935 NP_001310603.1 104 box-binding homeobox 1 isoform hZEB1 zinc finger E- mRNA ID: 6935 NM_001323675.2 105 box-bindinghomeobox 1 isoform i ZEB1 zinc finger E- protein ID: 6935 NP_001310604.1106 box-binding homeobox 1 isoform i ZEB1 zinc finger E- mRNA ID: 6935NM_001323676.2 107 box-binding homeobox 1 isoform j ZEB1 zinc finger E-protein ID: 6935 NP_001310605.1 108 box-binding homeobox 1 isoform jZEB1 zinc finger E- mRNA ID: 6935 NM_001323677.2 109 box-bindinghomeobox 1 isoform k ZEB1 zinc finger E- protein ID: 6935 NP_001310606.1110 box-binding homeobox 1 isoform k ZEB1 zinc finger E- mRNA ID: 6935NM_001323678.2 ill box-binding homeobox 1 isoform 1 ZEB1 zinc finger E-Protein ID: 6935 NP_001310607.1 112 box-binding homeobox 1 isoform 1ZEB1 zinc finger E- mRNA ID: 6935 NM_030751.6 113 box-binding homeobox 1isoform b ZEB1 zinc finger E- protein ID: 6935 NP_110378.3 114box-binding homeobox 1 isoform b ZEB1 zinc finger E- mRNA ID: 6935XM_006717498.2 115 box-binding homeobox 1 isoform X1 ZEB1 zinc finger E-protein ID: 6935 XP_006717561.1 116 box-binding homeobox 1 isoform X1ZEB1 zinc finger E- mRNA ID: 6935 XM_017016597.1 117 box-bindinghomeobox 1 isoform X1 ZEB1 zinc finger E- Protein ID: 6935XP_016872086.1 118 box-binding homeobox 1 isoform X1 ZEB1 zinc finger E-mRNA ID: 6935 XM_011519643.2 119 box-binding homeobox 1 isoform X1 ZEB1zinc finger E- Protein ID: 6935 XP_011517945.1 120 box-bindinghomeobox 1 isoform X1 AHNAK neuroblast mRNA ID: 79026 NM_001346445.2 121differentiation- associated protein AHNAK isoform 1 AHNAK neuroblastProtein ID: 79026 NP_001333374.1 122 differentiation- associated proteinAHNAK isoform 1 AHNAK neuroblast mRNA ID: 79026 NM_001346446.2 123differentiation- associated protein AHNAK isoform 1 AHNAK neuroblastProtein ID: 79026 NP_001333375.1 124 differentiation- associated proteinAHNAK isoform 1 AHNAK neuroblast mRNA ID: 79026 NM_001620.3 125differentiation- associated protein AHNAK isoform 1 AHNAK neuroblastProtein ID: 79026 NP_001611.1 126 differentiation- associated proteinAHNAK isoform 1 AHNAK neuroblast mRNA ID: 79026 NM_024060.4 127differentiation- associated protein AHNAK isoform 2 AHNAK neuroblastProtein ID: 79026 NP_076965.2 128 differentiation- associated proteinAHNAK isoform 2 AHNAK neuroblast mRNA ID: 79026 XM_017018270.1 129differentiation- associated protein AHNAK isoform X1 AHNAK neuroblastProtein ID: 79026 XP_016873759.1 130 differentiation- associated proteinAHNAK isoform X1

As described herein, levels of certain genes described herein, e.g., inexosomes, can be increased or decreased in subjects with cancer,subjects with diabetes or obesity, or in subjects at greater risk of EMTand/or metastasis. Accordingly, in one aspect of any of the embodiments,described herein is a method of treating cancer in a subject in needthereof, the method comprising a) administering a glucose-controllingmedication or obesity medication and/or b) administering CT scans at afrequency of higher than 1 CT scan every 6 months, to a subjectdetermined to have an increased level of expression of at least one geneselected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),E-cadherin (CDH1), ZEB1, and AHNAK; or a decreased level of expressionof at least one gene selected from: miR424-5p, miR-326, miR424-5p,miR-27a-3p, miR320b and miR320d; relative to a reference. In one aspectof any of the embodiments, described herein is a method of treatingcancer in a subject in need thereof, the method comprising: a)determining the level of expression of at least one gene selected from:miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail(SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin(CDH1), ZEB1, AHNAK, miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320band miR320d in a sample obtained from a subject; and b) i) administeringa glucose-controlling medication or obesity medication and/or ii)administering CT scans at a frequency of higher than 1 CT scan every 6months, to the subject if the expression level of at least one geneselected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),E-cadherin (CDH1), ZEB1, and AHNAK is increased relative to a reference;or if the expression level of at least one gene selected from:miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d; isdecreased relative to a reference.

in one aspect of any of the embodiments, described herein is a method oftreating cancer in a subject in need thereof, the method comprising a)i)administering a glucose-controlling medication or obesity medicationand/or ii) administering CT scans at a frequency of higher than 1 CTscan every 6 months, to a subject determined to have an increased levelof expression of at least one gene selected from: miR374a-5p, miR-93-5p,miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist (TWIST1),Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, and AHNAK; or adecreased level of expression of at least one gene selected from:miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d; relativeto a reference; or b) i) not administering a glucose-controllingmedication or obesity medication and/or ii) administering CT scans at afrequency of no more than 1 CT scan every 6 months, to a subjectdetermined not to have an increased level of expression of at least onegene selected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p,miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin(VIM), E-cadherin (CDH1), ZEB1, and AHNAK; or a decreased level ofexpression of at least one gene selected from: miR424-5p, miR-326,miR424-5p, miR-27a-3p, miR320b and miR320d; relative to a reference. Inone aspect of any of the embodiments, described herein is a method oftreating cancer in a subject in need thereof, the method comprising: a)determining the level of expression of at least one gene selected from:miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail(SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin(CDH1), ZEB1, AHNAK, miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320band miR320d in a sample obtained from a subject; b) i) administering aglucose-controlling medication or obesity medication and/or ii)administering CT scans at a frequency of higher than 1 CT scan every 6months, to the subject if the expression level of at least one geneselected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),E-cadherin (CDH1), ZEB1, and AHNAK is increased relative to a reference;or if the expression level of at least one gene selected from:miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d; isdecreased relative to a reference; and c) i) not administering aglucose-controlling medication or obesity medication and/or ii)administering CT scans at a frequency of no more than 1 CT scan every 6months, to the subject if the expression level of at least one geneselected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),E-cadherin (CDH1), ZEB1, and AHNAK is increased relative to a reference;or if the expression level of at least one gene selected from:miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d; isdecreased relative to a reference.

In some embodiments of any of the aspects, the method comprises a)administering a glucose-controlling medication or obesity medicationand/or b) administering CT scans at a frequency of higher than 1 CT scanevery 6 months, to a subject previously determined to have an increasedexpression level of at least one gene selected from: miR374a-5p,miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist(TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, andAHNAK; or a decreased level of expression of at least one gene selectedfrom: miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d;relative to a reference. In some embodiments of any of the aspects,described herein is a method of treating cancer in a subject in needthereof, the method comprising: a) first determining the level ofexpression of at least one gene selected from: miR374a-5p, miR-93-5p,miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist (TWIST1),Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, AHNAK, miR424-5p,miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d in a sample obtainedfrom a subject; and b) then i) administering a glucose-controllingmedication or obesity medication and/or ii) administering CT scans at afrequency of higher than 1 CT scan every 6 months, to the subject if theexpression level of at least one gene selected from: miR374a-5p,miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist(TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, andAHNAK is increased relative to a reference; or if the expression levelof at least one gene selected from: miR424-5p, miR-326, miR424-5p,miR-27a-3p, miR320b and miR320d; is decreased relative to a reference.

In some embodiments of any of the aspects, the method comprises a) i)administering a glucose-controlling medication or obesity medicationand/or ii) administering CT scans at a frequency of higher than 1 CTscan every 6 months, to a subject previously determined to have anincreased expression level of at least one gene selected from:miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail(SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin(CDH1), ZEB1, and AHNAK; or a decreased level of expression of at leastone gene selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p,miR320b and miR320d; relative to a reference; and b) i) notadministering a glucose-controlling medication or obesity medicationand/or ii) administering CT scans at a frequency of no more than 1 CTscan every 6 months, to a subject previously determined to not have anincreased expression level of at least one gene selected from:miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail(SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin(CDH1), ZEB1, and AHNAK; or a decreased level of expression of at leastone gene selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p,miR320b and miR320d; relative to a reference. In some embodiments of anyof the aspects, described herein is a method of treating cancer in asubject in need thereof, the method comprising: a) first determining thelevel of expression of at least one gene selected from: miR374a-5p,miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist(TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, AHNAK,miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d in asample obtained from a subject; and then b) i) administering aglucose-controlling medication or obesity medication and/or ii)administering CT scans at a frequency of higher than 1 CT scan every 6months, to the subject if the expression level of at least one geneselected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),E-cadherin (CDH1), ZEB1, and AHNAK is increased relative to a reference;or if the expression level of at least one gene selected from:miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d; isdecreased relative to a reference; or c) i) not administering aglucose-controlling medication or obesity medication and/or ii)administering CT scans at a frequency of no more than 1 CT scan every 6months, to the subject if the expression level of at least one geneselected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),E-cadherin (CDH1), ZEB1, and AHNAK is increased relative to a reference;or if the expression level of at least one gene selected from:miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d; isdecreased relative to a reference.

In one aspect of any of the embodiments, described herein is a method oftreating cancer in a subject in need thereof, the method comprising: a)determining if the subject has an expression level of at least one geneselected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),E-cadherin (CDH1), ZEB1, and AHNAK that is increased relative to areference; or an expression level of at least one gene selected from:miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d; that isdecreased relative to a reference; and b) i) administering aglucose-controlling medication or obesity medication and/or ii)administering CT scans at a frequency of higher than 1 CT scan every 6months, to the subject if the expression level of at least one geneselected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),E-cadherin (CDH1), ZEB1, and AHNAK is increased relative to a reference;or if the expression level of at least one gene selected from:miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d; isdecreased relative to a reference. In one aspect of any of theembodiments, described herein is a method of treating cancer in asubject in need thereof, the method comprising: a) determining if thesubject has an expression level of at least one gene selected from:miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail(SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin(CDH1), ZEB1, and AHNAK that is increased relative to a reference; or anexpression level of at least one gene selected from: miR424-5p, miR-326,miR424-5p, miR-27a-3p, miR320b and miR320d; that is decreased relativeto a reference; and b) i) administering a glucose-controlling medicationor obesity medication and/or ii) administering CT scans at a frequencyof higher than 1 CT scan every 6 months, to the subject if theexpression level of at least one gene selected from: miR374a-5p,miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist(TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, andAHNAK is increased relative to a reference; or if the expression levelof at least one gene selected from: miR424-5p, miR-326, miR424-5p,miR-27a-3p, miR320b and miR320d; is decreased relative to a reference;or c) i) not administering a glucose-controlling medication or obesitymedication and/or ii) administering CT scans at a frequency of no morethan 1 CT scan every 6 months, to the subject if the expression level ofat least one gene selected from: miR374a-5p, miR-93-5p, miR-28-3p,miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug(SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, and AHNAK is increasedrelative to a reference; or if the expression level of at least one geneselected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b andmiR320d; is decreased relative to a reference. In some embodiments ofany of the aspects, the step of determining if the subject has anincreased expression level of at least one gene selected from:miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail(SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin(CDH1), ZEB1, and AHNAK; or a decreased level of expression of at leastone gene selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p,miR320b and miR320d; can comprise i) obtaining or having obtained asample from the subject and ii) performing or having performed an assayon the sample obtained from the subject to determine/measure the levelof at least one of the foregoing genes in the subject. In someembodiments of any of the aspects, the step of determining if thesubject has an increased expression level of at least one gene selectedfrom: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5,Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin(CDH1), ZEB1, and AHNAK; or a decreased level of expression of at leastone gene selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p,miR320b and miR320d; can comprise performing or having performed anassay on a sample obtained from the subject to determine/measure thelevel of at least one of the foregoing genes in the subject. In someembodiments of any of the aspects, the step of determining if thesubject has an increased expression level of at least one gene selectedfrom: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5,Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin(CDH1), ZEB1, and AHNAK; or a decreased level of expression of at leastone gene selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p,miR320b and miR320d; can comprise ordering or requesting an assay on asample obtained from the subject to determine/measure the level of atleast one of the foregoing genes in the subject. In some embodiments ofany of the aspects, the step of determining if the subject has anincreased expression level of at least one gene selected from:miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail(SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin(CDH1), ZEB1, and AHNAK; or a decreased level of expression of at leastone gene selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p,miR320b and miR320d; can comprise receiving the results of an assay on asample obtained from the subject to determine/measure the level of atleast one of the foregoing genes in the subject. In some embodiments ofany of the aspects, the step of determining if the subject has anincreased expression level of at least one gene selected from:miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail(SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin(CDH1), ZEB1, and AHNAK; or a decreased level of expression of at leastone gene selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p,miR320b and miR320d; can comprise receiving a report, results, or othermeans of identifying the subject as a subject with an increased and/ordecreased level of expression of at least one of the foregoing genes.

In one aspect of any of the embodiments, described herein is a method oftreating cancer in a subject in need thereof, the method comprising: a)determining if the subject has an increased expression level of at leastone gene selected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p,miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin(VIM), E-cadherin (CDH1), ZEB1, and AHNAK; or a decreased level ofexpression of at least one gene selected from: miR424-5p, miR-326,miR424-5p, miR-27a-3p, miR320b and miR320d; and b) instructing ordirecting that the subject be i) administering a glucose-controllingmedication or obesity medication and/or ii) administering CT scans at afrequency of higher than 1 CT scan every 6 months, if the expressionlevel of at least one gene selected from: miR374a-5p, miR-93-5p,miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist (TWIST1),Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, and AHNAK isincreased relative to a reference; or if the expression level of atleast one gene selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p,miR320b and miR320d; is decreased relative to a reference. In one aspectof any of the embodiments, described herein is a method of treatingcancer in a subject in need thereof, the method comprising: a)determining if the subject has an increased expression level of at leastone gene selected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p,miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin(VIM), E-cadherin (CDH1), ZEB1, and AHNAK; or a decreased level ofexpression of at least one gene selected from: miR424-5p, miR-326,miR424-5p, miR-27a-3p, miR320b and miR320d; and b) instructing ordirecting that the subject be i) administered a glucose-controllingmedication or obesity medication and/or ii) administered CT scans at afrequency of higher than 1 CT scan every 6 months, if the expressionlevel of at least one gene selected from: miR374a-5p, miR-93-5p,miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist (TWIST1),Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, and AHNAK isincreased relative to a reference; or if the expression level of atleast one gene selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p,miR320b and miR320d; is decreased relative to a reference; or c)instructing or directing that the subject be i) not administered aglucose-controlling medication or obesity medication and/or ii)administered CT scans at a frequency of no more than 1 CT scan every 6months, if the expression level of at least one gene selected from:miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail(SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin(CDH1), ZEB1, and AHNAK is increased relative to a reference; or if theexpression level of at least one gene selected from: miR424-5p, miR-326,miR424-5p, miR-27a-3p, miR320b and miR320d; is decreased relative to areference. In some embodiments of any of the aspects, the step ofdetermining if the subject has an increased expression level of at leastone gene selected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p,miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin(VIM), E-cadherin (CDH1), ZEB1, and AHNAK; or a decreased level ofexpression of at least one gene selected from: miR424-5p, miR-326,miR424-5p, miR-27a-3p, miR320b and miR320d; can comprise i) obtaining orhaving obtained a sample from the subject and ii) performing or havingperformed an assay on the sample obtained from the subject todetermine/measure the level of at least one of the foregoing genes inthe subject. In some embodiments of any of the aspects, the step ofdetermining if the subject has an increased expression level of at leastone gene selected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p,miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin(VIM), E-cadherin (CDH1), ZEB1, and AHNAK; or a decreased level ofexpression of at least one gene selected from: miR424-5p, miR-326,miR424-5p, miR-27a-3p, miR320b and miR320d; can comprise performing orhaving performed an assay on a sample obtained from the subject todetermine/measure the level of at least one of the foregoing genes inthe subject. In some embodiments of any of the aspects, the step ofdetermining if the subject has an increased expression level of at leastone gene selected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p,miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin(VIM), E-cadherin (CDH1), ZEB1, and AHNAK; or a decreased level ofexpression of at least one gene selected from: miR424-5p, miR-326,miR424-5p, miR-27a-3p, miR320b and miR320d; can comprise ordering orrequesting an assay on a sample obtained from the subject todetermine/measure the level of at least one of the foregoing genes inthe subject. In some embodiments of any of the aspects, the step ofinstructing or directing that the subject be administered a particulartreatment can comprise providing a report of the assay results. In someembodiments of any of the aspects, the step of instructing or directingthat the subject be administered a particular treatment can compriseproviding a report of the assay results and/or treatment recommendationsin view of the assay results.

In one aspect of any of the embodiments, described herein is a method ofdetermining if a subject has cancer, increased risk of cancer, a highlevel of EMT or metastasis, or is at increased risk of EMT ormetastasis, or is in need of treatment for cancer, EMT, or metastasis,the method comprising: determining the level of expression of at leastone gene selected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p,miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin(VIM), E-cadherin (CDH1), ZEB1, AHNAK, miR424-5p, miR-326, miR424-5p,miR-27a-3p, miR320b and miR320d in a sample obtained from the subject,wherein an increased expression level of at least one gene selectedfrom: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5,Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin(CDH1), ZEB1, and AHNAK; or a decreased level of expression of at leastone gene selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p,miR320b and miR320d; relative to a reference indicates the subject hascancer, increased risk of cancer, a high level of EMT or metastasis, oris at increased risk of EMT or metastasis, or is in need of treatmentfor cancer, EMT, or metastasis.

In some embodiments of any of the aspects, the glucose-controllingmedication is metformin, a sulfonylurea (e.g., glyburide, glipizide, orglimepiride), a glinide, a SGLT2 inhibitor (e.g., canagliflozin,dapagliflozin, or empagliflozin), or insulin. In some embodiments of anyof the aspects, the glucose-controlling medication is metformin. In someembodiments of any of the aspects, the obesity medication is orlistat,phentermine-topiramate, naltrexone-bupropion, liraglutide, semagludtide,setmelanotide, phentermine, benzphetamine, diethylpropion, orphendimetrazine.

In some embodiments of any of the aspects, administering CT scans at afrequency of higher than 1 CT scan every 6 months comprisesadministering more than 1 CT scan every 6 months for at least one year.In some embodiments of any of the aspects, administering CT scans at afrequency of higher than 1 CT scan every 6 months comprisesadministering more than 1 CT scan every 6 months for at least two years.In some embodiments of any of the aspects, administering CT scans at afrequency of higher than 1 CT scan every 6 months comprisesadministering more than 1 CT scan every 6 months for at least threeyears.

In some embodiments of any of the aspects, administering CT scans at afrequency of higher than 1 CT scan every 6 months comprisesadministering at least one CT scan every 4 months for at least one year.In some embodiments of any of the aspects, administering CT scans at afrequency of higher than 1 CT scan every 6 months comprisesadministering at least one CT scan every 4 months for at least twoyears. In some embodiments of any of the aspects, administering CT scansat a frequency of higher than 1 CT scan every 6 months comprisesadministering at least one CT scan every 4 months for at least threeyears.

In some embodiments of any of the aspects, administering CT scans at afrequency of higher than 1 CT scan every 6 months comprisesadministering at least one CT scan every 3 months for at least one year.In some embodiments of any of the aspects, administering CT scans at afrequency of higher than 1 CT scan every 6 months comprisesadministering at least one CT scan every 3 months for at least twoyears. In some embodiments of any of the aspects, administering CT scansat a frequency of higher than 1 CT scan every 6 months comprisesadministering at least one CT scan every 3 months for at least threeyears.

In some embodiments of any of the aspects, administering CT scans at afrequency of no more than 1 CT scan every 6 months comprisesadministering one CT scan every 6 months for at least one year. In someembodiments of any of the aspects, administering CT scans at a frequencyof no more than 1 CT scan every 6 months comprises administering one CTscan every 6 months for at least two years. In some embodiments of anyof the aspects, administering CT scans at a frequency of no more than 1CT scan every 6 months comprises administering one CT scan every 6months for at least three years.

In some embodiments of any of the aspects, administering CT scans at afrequency of no more than 1 CT scan every 6 months comprisesadministering one CT scan year for at least one year. In someembodiments of any of the aspects, administering CT scans at a frequencyof no more than 1 CT scan every 6 months comprises administering one CTscan every year for at least two years. In some embodiments of any ofthe aspects, administering CT scans at a frequency of no more than 1 CTscan every 6 months comprises administering one CT scan every year forat least three years.

In some embodiments of any of the aspects, measurement of the level of atarget and/or detection of the level or presence of a target, e.g. of anexpression product (nucleic acid or polypeptide of one of the genesdescribed herein) or a mutation can comprise a transformation. As usedherein, the term “transforming” or “transformation” refers to changingan object or a substance, e.g., biological sample, nucleic acid orprotein, into another substance. The transformation can be physical,biological or chemical. Exemplary physical transformation includes, butis not limited to, pre-treatment of a biological sample, e.g., fromwhole blood to blood serum by differential centrifugation. Abiological/chemical transformation can involve the action of at leastone enzyme and/or a chemical reagent in a reaction. For example, a DNAsample can be digested into fragments by one or more restrictionenzymes, or an exogenous molecule can be attached to a fragmented DNAsample with a ligase. In some embodiments of any of the aspects, a DNAsample can undergo enzymatic replication, e.g., by polymerase chainreaction (PCR).

Transformation, measurement, and/or detection of a target molecule, e.g.a mRNA or polypeptide can comprise contacting a sample obtained from asubject with a reagent (e.g. a detection reagent) which is specific forthe target, e.g., a target-specific reagent. In some embodiments of anyof the aspects, the target-specific reagent is detectably labeled. Insome embodiments of any of the aspects, the target-specific reagent iscapable of generating a detectable signal. In some embodiments of any ofthe aspects, the target-specific reagent generates a detectable signalwhen the target molecule is present.

Methods to measure gene expression products are known to a skilledartisan. Such methods to measure gene expression products, e.g., proteinlevel, include ELISA (enzyme linked immunosorbent assay), western blot,immunoprecipitation, and immunofluorescence using detection reagentssuch as an antibody or protein binding agents. Alternatively, a peptidecan be detected in a subject by introducing into a subject a labeledanti-peptide antibody and other types of detection agent. For example,the antibody can be labeled with a detectable marker whose presence andlocation in the subject is detected by standard imaging techniques.

For example, antibodies for the various targets described herein arecommercially available and can be used for the purposes of the inventionto measure protein expression levels. Alternatively, since the aminoacid sequences for the targets described herein are known and publicallyavailable at the NCBI website, one of skill in the art can raise theirown antibodies against these polypeptides of interest for the purpose ofthe methods described herein.

The amino acid sequences of the polypeptides described herein have beenassigned NCBI accession numbers for different species such as human,mouse and rat.

In some embodiments of any of the aspects, immunohistochemistry (“IHC”)and immunocytochemistry (“ICC”) techniques can be used. IHC is theapplication of immunochemistry to tissue sections, whereas ICC is theapplication of immunochemistry to cells or tissue imprints after theyhave undergone specific cytological preparations such as, for example,liquid-based preparations. Immunochemistry is a family of techniquesbased on the use of an antibody, wherein the antibodies are used tospecifically target molecules inside or on the surface of cells. Theantibody typically contains a marker that will undergo a biochemicalreaction, and thereby experience a change of color, upon encounteringthe targeted molecules. In some instances, signal amplification can beintegrated into the particular protocol, wherein a secondary antibody,that includes the marker stain or marker signal, follows the applicationof a primary specific antibody.

In some embodiments of any of the aspects, the assay can be a Westernblot analysis. Alternatively, proteins can be separated bytwo-dimensional gel electrophoresis systems. Two-dimensional gelelectrophoresis is well known in the art and typically involvesiso-electric focusing along a first dimension followed by SDS-PAGEelectrophoresis along a second dimension. These methods also require aconsiderable amount of cellular material. The analysis of 2D SDS-PAGEgels can be performed by determining the intensity of protein spots onthe gel, or can be performed using immune detection. In otherembodiments, protein samples are analyzed by mass spectroscopy.

Immunological tests can be used with the methods and assays describedherein and include, for example, competitive and non-competitive assaysystems using techniques such as Western blots, radioimmunoassay (RIA),ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, immunodiffusion assays, agglutinationassays, e.g. latex agglutination, complement-fixation assays,immunoradiometric assays, fluorescent immunoassays, e.g. FIA(fluorescence-linked immunoassay), chemiluminescence immunoassays(CLIA), electrochemiluminescence immunoassay (ECLIA, countingimmunoassay (CIA), lateral flow tests or immunoassay (LFIA), magneticimmunoassay (MIA), and protein A immunoassays. Methods for performingsuch assays are known in the art, provided an appropriate antibodyreagent is available. In some embodiments of any of the aspects, theimmunoassay can be a quantitative or a semi-quantitative immunoassay.

An immunoassay is a biochemical test that measures the concentration ofa substance in a biological sample, typically a fluid sample such asblood or serum, using the interaction of an antibody or antibodies toits antigen. The assay takes advantage of the highly specific binding ofan antibody with its antigen. For the methods and assays describedherein, specific binding of the target polypeptides with respectiveproteins or protein fragments, or an isolated peptide, or a fusionprotein described herein occurs in the immunoassay to form a targetprotein/peptide complex. The complex is then detected by a variety ofmethods known in the art. An immunoassay also often involves the use ofa detection antibody.

Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassayor EIA, is a biochemical technique used mainly in immunology to detectthe presence of an antibody or an antigen in a sample. The ELISA hasbeen used as a diagnostic tool in medicine and plant pathology, as wellas a quality control check in various industries.

In one embodiment, an ELISA involving at least one antibody withspecificity for the particular desired antigen (e.g., any of the targetsas described herein) can also be performed. A known amount of sampleand/or antigen is immobilized on a solid support (usually a polystyrenemicro titer plate). Immobilization can be either non-specific (e.g., byadsorption to the surface) or specific (e.g. where another antibodyimmobilized on the surface is used to capture antigen or a primaryantibody). After the antigen is immobilized, the detection antibody isadded, forming a complex with the antigen. The detection antibody can becovalently linked to an enzyme, or can itself be detected by a secondaryantibody which is linked to an enzyme through bio-conjugation. Betweeneach step the plate is typically washed with a mild detergent solutionto remove any proteins or antibodies that are not specifically bound.After the final wash step the plate is developed by adding an enzymaticsubstrate to produce a visible signal, which indicates the quantity ofantigen in the sample. Older ELISAs utilize chromogenic substrates,though newer assays employ fluorogenic substrates with much highersensitivity.

In another embodiment, a competitive ELISA is used. Purified antibodiesthat are directed against a target polypeptide or fragment thereof arecoated on the solid phase of multi-well plate, i.e., conjugated to asolid surface. A second batch of purified antibodies that are notconjugated on any solid support is also needed. These non-conjugatedpurified antibodies are labeled for detection purposes, for example,labeled with horseradish peroxidase to produce a detectable signal. Asample (e.g., a blood sample) from a subject is mixed with a knownamount of desired antigen (e.g., a known volume or concentration of asample comprising a target polypeptide) together with the horseradishperoxidase labeled antibodies and the mixture is then are added tocoated wells to form competitive combination. After incubation, if thepolypeptide level is high in the sample, a complex of labeled antibodyreagent-antigen will form. This complex is free in solution and can bewashed away. Washing the wells will remove the complex. Then the wellsare incubated with TMB (3, 3′, 5, 5′-tetramethylbenzidene) colordevelopment substrate for localization of horseradishperoxidase-conjugated antibodies in the wells. There will be no colorchange or little color change if the target polypeptide level is high inthe sample. If there is little or no target polypeptide present in thesample, a different complex in formed, the complex of solid supportbound antibody reagents-target polypeptide. This complex is immobilizedon the plate and is not washed away in the wash step. Subsequentincubation with TMB will produce significant color change. Such acompetitive ELSA test is specific, sensitive, reproducible and easy tooperate.

There are other different forms of ELISA, which are well known to thoseskilled in the art. The standard techniques known in the art for ELISAare described in “Methods in Immunodiagnosis”, 2nd Edition, Rose andBigazzi, eds. John Wiley & Sons, 1980; and Oellerich, M. 1984, J. Clin.Chem. Clin. Biochem. 22:895-904. These references are herebyincorporated by reference in their entirety.

In one embodiment, the levels of a polypeptide in a sample can bedetected by a lateral flow immunoassay test (LFIA), also known as theimmunochromatographic assay, or strip test. LFIAs are a simple deviceintended to detect the presence (or absence) of antigen, e.g. apolypeptide, in a fluid sample. There are currently many LFIA tests usedfor medical diagnostics, either for home testing, point of care testing,or laboratory use. LFIA tests are a form of immunoassay in which thetest sample flows along a solid substrate via capillary action. Afterthe sample is applied to the test strip it encounters a colored reagent(generally comprising antibody specific for the test target antigen)bound to microparticles which mixes with the sample and transits thesubstrate encountering lines or zones which have been pretreated withanother antibody or antigen. Depending upon the level of targetpolypeptides present in the sample the colored reagent can be capturedand become bound at the test line or zone. LFIAs are essentiallyimmunoassays adapted to operate along a single axis to suit the teststrip format or a dipstick format. Strip tests are extremely versatileand can be easily modified by one skilled in the art for detecting anenormous range of antigens from fluid samples such as urine, blood,water, and/or homogenized tissue samples etc. Strip tests are also knownas dip stick tests, the name bearing from the literal action of“dipping” the test strip into a fluid sample to be tested. LFIA striptests are easy to use, require minimum training and can easily beincluded as components of point-of-care test (POCT) diagnostics to beuse on site in the field. LFIA tests can be operated as eithercompetitive or sandwich assays. Sandwich LFIAs are similar to sandwichELISA. The sample first encounters colored particles which are labeledwith antibodies raised to the target antigen. The test line will alsocontain antibodies to the same target, although it may bind to adifferent epitope on the antigen. The test line will show as a coloredband in positive samples. In some embodiments of any of the aspects, thelateral flow immunoassay can be a double antibody sandwich assay, acompetitive assay, a quantitative assay or variations thereof.Competitive LFIAs are similar to competitive ELISA. The sample firstencounters colored particles which are labeled with the target antigenor an analogue. The test line contains antibodies to the target/itsanalogue. Unlabelled antigen in the sample will block the binding siteson the antibodies preventing uptake of the colored particles. The testline will show as a colored band in negative samples. There are a numberof variations on lateral flow technology. It is also possible to applymultiple capture zones to create a multiplex test.

The use of “dip sticks” or LFIA test strips and other solid supportshave been described in the art in the context of an immunoassay for anumber of antigen biomarkers. U.S. Pat. Nos. 4,943,522; 6,485,982;6,187,598; 5,770,460; 5,622,871; 6,565,808, U.S. patent applicationsSer. No. 10/278,676; U.S. Ser. No. 09/579,673 and U.S. Ser. No.10/717,082, which are incorporated herein by reference in theirentirety, are non-limiting examples of such lateral flow test devices.Examples of patents that describe the use of “dip stick” technology todetect soluble antigens via immunochemical assays include, but are notlimited to U.S. Pat. Nos. 4,444,880; 4,305,924; and 4,135,884; which areincorporated by reference herein in their entireties. The apparatusesand methods of these three patents broadly describe a first componentfixed to a solid surface on a “dip stick” which is exposed to a solutioncontaining a soluble antigen that binds to the component fixed upon the“dip stick,” prior to detection of the component-antigen complex uponthe stick. It is within the skill of one in the art to modify theteachings of this “dip stick” technology for the detection ofpolypeptides using antibody reagents as described herein.

Other techniques can be used to detect the level of a polypeptide in asample. One such technique is the dot blot, an adaptation of Westernblotting (Towbin et at., Proc. Nat. Acad. Sci. 76:4350 (1979)). In aWestern blot, the polypeptide or fragment thereof can be dissociatedwith detergents and heat, and separated on an SDS-PAGE gel before beingtransferred to a solid support, such as a nitrocellulose or PVDFmembrane. The membrane is incubated with an antibody reagent specificfor the target polypeptide or a fragment thereof The membrane is thenwashed to remove unbound proteins and proteins with non-specificbinding. Detectably labeled enzyme-linked secondary or detectionantibodies can then be used to detect and assess the amount ofpolypeptide in the sample tested. A dot blot immobilizes a proteinsample on a defined region of a support, which is then probed withantibody and labelled secondary antibody as in Western blotting. Theintensity of the signal from the detectable label in either formatcorresponds to the amount of enzyme present, and therefore the amount ofpolypeptide. Levels can be quantified, for example by densitometry.

In some embodiments of any of the aspects, the level of a target can bemeasured, by way of non-limiting example, by Western blot;immunoprecipitation; enzyme-linked immunosorbent assay (ELISA);radioimmunological assay (RIA); sandwich assay; fluorescence in situhybridization (FISH); immunohistological staining; radioimmunometricassay; immunofluoresence assay; mass spectroscopy and/orimmunoelectrophoresis assay.

In certain embodiments, the gene expression products as described hereincan be instead determined by determining the level of messenger RNA(mRNA) expression of the genes described herein. Such molecules can beisolated, derived, or amplified from a biological sample, such as ablood sample. Techniques for the detection of mRNA expression is knownby persons skilled in the art, and can include but not limited to, PCRprocedures, RT-PCR, quantitative RT-PCR Northern blot analysis,differential gene expression, RNAse protection assay, microarray basedanalysis, next-generation sequencing; hybridization methods, etc.

In general, the PCR procedure describes a method of gene amplificationwhich is comprised of (i) sequence-specific hybridization of primers tospecific genes or sequences within a nucleic acid sample or library,(ii) subsequent amplification involving multiple rounds of annealing,elongation, and denaturation using a thermostable DNA polymerase, and(iii) screening the PCR products for a band of the correct size. Theprimers used are oligonucleotides of sufficient length and appropriatesequence to provide initiation of polymerization, i.e. each primer isspecifically designed to be complementary to a strand of the genomiclocus to be amplified. In an alternative embodiment, mRNA level of geneexpression products described herein can be determined byreverse-transcription (RT) PCR and by quantitative RT-PCR (QRT-PCR) orreal-time PCR methods. Methods of RT-PCR and QRT-PCR are well known inthe art.

In some embodiments of any of the aspects, the level of an mRNA can bemeasured by a quantitative sequencing technology, e.g. a quantitativenext-generation sequence technology. Methods of sequencing a nucleicacid sequence are well known in the art. Briefly, a sample obtained froma subject can be contacted with one or more primers which specificallyhybridize to a single-strand nucleic acid sequence flanking the targetgene sequence and a complementary strand is synthesized. In somenext-generation technologies, an adaptor (double or single-stranded) isligated to nucleic acid molecules in the sample and synthesis proceedsfrom the adaptor or adaptor compatible primers. In some third-generationtechnologies, the sequence can be determined, e.g. by determining thelocation and pattern of the hybridization of probes, or measuring one ormore characteristics of a single molecule as it passes through a sensor(e.g. the modulation of an electrical field as a nucleic acid moleculepasses through a nanopore). Exemplary methods of sequencing include, butare not limited to, Sanger sequencing, dideoxy chain termination,high-throughput sequencing, next generation sequencing, 454 sequencing,SOLiD sequencing, polony sequencing, Illumina sequencing, Ion Torrentsequencing, sequencing by hybridization, nanopore sequencing, Helioscopesequencing, single molecule real time sequencing, RNAP sequencing, andthe like. Methods and protocols for performing these sequencing methodsare known in the art, see, e.g. “Next Generation Genome Sequencing” Ed.Michal Janitz, Wiley-VCH; “High-Throughput Next Generation Sequencing”Eds. Kwon and Ricke, Humanna Press, 2011; and Sambrook et al., MolecularCloning: A Laboratory Manual (4 ed.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA (2012); which are incorporated byreference herein in their entireties.

The nucleic acid sequences of the genes described herein have beenassigned NCBI accession numbers for different species such as human,mouse and rat. Accordingly, a skilled artisan can design an appropriateprimer based on the known sequence for determining the mRNA level of therespective gene.

Nucleic acid and ribonucleic acid (RNA) molecules can be isolated from aparticular biological sample using any of a number of procedures, whichare well-known in the art, the particular isolation procedure chosenbeing appropriate for the particular biological sample. For example,freeze-thaw and alkaline lysis procedures can be useful for obtainingnucleic acid molecules from solid materials; heat and alkaline lysisprocedures can be useful for obtaining nucleic acid molecules fromurine; and proteinase K extraction can be used to obtain nucleic acidfrom blood (Roiff, A et al. PCR: Clinical Diagnostics and Research,Springer (1994)).

In some embodiments of any of the aspects, one or more of the reagents(e.g. an antibody reagent and/or nucleic acid probe) described hereincan comprise a detectable label and/or comprise the ability to generatea detectable signal (e.g. by catalyzing reaction converting a compoundto a detectable product). Detectable labels can comprise, for example, alight-absorbing dye, a fluorescent dye, or a radioactive label.Detectable labels, methods of detecting them, and methods ofincorporating them into reagents (e.g. antibodies and nucleic acidprobes) are well known in the art.

In some embodiments of any of the aspects, detectable labels can includelabels that can be detected by spectroscopic, photochemical,biochemical, immunochemical, electromagnetic, radiochemical, or chemicalmeans, such as fluorescence, chemifluoresence, or chemiluminescence, orany other appropriate means. The detectable labels used in the methodsdescribed herein can be primary labels (where the label comprises amoiety that is directly detectable or that produces a directlydetectable moiety) or secondary labels (where the detectable label bindsto another moiety to produce a detectable signal, e.g., as is common inimmunological labeling using secondary and tertiary antibodies). Thedetectable label can be linked by covalent or non-covalent means to thereagent. Alternatively, a detectable label can be linked such as bydirectly labeling a molecule that achieves binding to the reagent via aligand-receptor binding pair arrangement or other such specificrecognition molecules. Detectable labels can include, but are notlimited to radioisotopes, bioluminescent compounds, chromophores,antibodies, chemiluminescent compounds, fluorescent compounds, metalchelates, and enzymes.

In other embodiments, the detection reagent is label with a fluorescentcompound. When the fluorescently labeled reagent is exposed to light ofthe proper wavelength, its presence can then be detected due tofluorescence. In some embodiments of any of the aspects, a detectablelabel can be a fluorescent dye molecule, or fluorophore including, butnot limited to fluorescein, phycoerythrin, phycocyanin, o-phthaldehyde,fluorescamine, Cy3™, Cy5™, allophycocyanine, Texas Red, perideninchlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5™,green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC)and Oregon Green™, rhodamine and derivatives (e.g., Texas red andtetrarhodimine isothiocynate (TRITC)), biotin, phycoerythrin, AMCA,CyDyes™, 6-carboxyfhiorescein (commonly known by the abbreviations FAMand F), 6-carboxy-2′,4′,7′,4,7-hexachlorofiuorescein (HEX),6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfiuorescein (JOE or J),N,N,N′,N′-tetramethyl-6carboxyrhodamine (TAMRA or T),6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5),6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes,e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimidedyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidiumdyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes;polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc; BODIPY dyesand quinoline dyes. In some embodiments of any of the aspects, adetectable label can be a radiolabel including, but not limited to ₃H,₁₂₅I, ₃₅S, ¹⁴C, ³²P, and ³³P. In some embodiments of any of the aspects,a detectable label can be an enzyme including, but not limited tohorseradish peroxidase and alkaline phosphatase. An enzymatic label canproduce, for example, a chemiluminescent signal, a color signal, or afluorescent signal. Enzymes contemplated for use to detectably label anantibody reagent include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-V-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-VI-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. In some embodiments of any of the aspects, adetectable label is a chemiluminescent label, including, but not limitedto lucigenin, luminol, luciferin, isoluminol, theromatic acridiniumester, imidazole, acridinium salt and oxalate ester. In some embodimentsof any of the aspects, a detectable label can be a spectral colorimetriclabel including, but not limited to colloidal gold or colored glass orplastic (e.g., polystyrene, polypropylene, and latex) beads.

In some embodiments of any of the aspects, detection reagents can alsobe labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG,V5, HIS, or biotin. Other detection systems can also be used, forexample, a biotin-streptavidin system. In this system, the antibodiesimmunoreactive (i. e. specific for) with the biomarker of interest isbiotinylated. Quantity of biotinylated antibody bound to the biomarkeris determined using a streptavidin-peroxidase conjugate and achromagenic substrate. Such streptavidin peroxidase detection kits arecommercially available, e. g. from DAKO; Carpinteria, Calif. A reagentcan also be detectably labeled using fluorescence emitting metals suchas ¹⁵²Eu, or others of the lanthanide series. These metals can beattached to the reagent using such metal chelating groups asdiethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetraaceticacid (EDTA).

A level which is less than a reference level can be a level which isless by at least about 10%, at least about 20%, at least about 50%, atleast about 60%, at least about 80%, at least about 90%, or lessrelative to the reference level. In some embodiments of any of theaspects, a level which is less than a reference level can be a levelwhich is statistically significantly less than the reference level.

A level which is more than a reference level can be a level which isgreater by at least about 10%, at least about 20%, at least about 50%,at least about 60%, at least about 80%, at least about 90%, at leastabout 100%, at least about 200%, at least about 300%, at least about500% or more than the reference level. In some embodiments of any of theaspects, a level which is more than a reference level can be a levelwhich is statistically significantly greater than the reference level.

In some embodiments of any of the aspects, the reference can be a levelof the target molecule in a population of subjects who do not have orare not diagnosed as having, and/or do not exhibit signs or symptoms ofcancer or diabetes. In some embodiments of any of the aspects, thereference can also be a level of expression of the target molecule in acontrol sample, a pooled sample of control individuals or a numericvalue or range of values based on the same. In some embodiments of anyof the aspects, the reference can be the level of a target molecule in asample obtained from the same subject at an earlier point in time, e.g.,the methods described herein can be used to determine if a subject'ssensitivity or response to a given therapy is changing over time.

In some embodiments of any of the aspects, the level of expressionproducts of no more than 200 other genes is determined. In someembodiments of any of the aspects, the level of expression products ofno more than 100 other genes is determined. In some embodiments of anyof the aspects, the level of expression products of no more than 20other genes is determined. In some embodiments of any of the aspects,the level of expression products of no more than 10 other genes isdetermined.

In some embodiments of the foregoing aspects, the expression level of agiven gene can be normalized relative to the expression level of one ormore reference genes or reference proteins.

In some embodiments, the reference level can be the level in a sample ofsimilar cell type, sample type, sample processing, and/or obtained froma subject of similar age, sex and other demographic parameters as thesample/subject for which the level of a gene described herein is to bedetermined. In some embodiments, the test sample and control referencesample are of the same type, that is, obtained from the same biologicalsource, and comprising the same composition, e.g. the same number andtype of cells.

The term “sample” or “test sample” as used herein denotes a sample takenor isolated from a biological organism, e.g., a blood or plasma samplefrom a subject. In some embodiments of any of the aspects, the presentinvention encompasses several examples of a biological sample. In someembodiments of any of the aspects, the biological sample is cells, ortissue, or peripheral blood, or bodily fluid. Exemplary biologicalsamples include, but are not limited to, a biopsy, a tumor sample,biofluid sample; blood; serum; plasma; urine; sperm; mucus; tissuebiopsy; organ biopsy; synovial fluid; bile fluid; cerebrospinal fluid;mucosal secretion; effusion; sweat; saliva; and/or tissue sample etc.The term also includes a mixture of the above-mentioned samples. Theterm “test sample” also includes untreated or pretreated (orpre-processed) biological samples. In some embodiments of any of theaspects, a test sample can comprise cells from a subject.

The test sample can be obtained by removing a sample from a subject, butcan also be accomplished by using a previously isolated sample (e.g.isolated at a prior timepoint and isolated by the same or anotherperson).

In some embodiments of any of the aspects, the test sample can be anuntreated test sample. As used herein, the phrase “untreated testsample” refers to a test sample that has not had any prior samplepre-treatment except for dilution and/or suspension in a solution.Exemplary methods for treating a test sample include, but are notlimited to, centrifugation, filtration, sonication, homogenization,heating, freezing and thawing, and combinations thereof. In someembodiments of any of the aspects, the test sample can be a frozen testsample, e.g., a frozen tissue. The frozen sample can be thawed beforeemploying methods, assays and systems described herein. After thawing, afrozen sample can be centrifuged before being subjected to methods,assays and systems described herein. In some embodiments of any of theaspects, the test sample is a clarified test sample, for example, bycentrifugation and collection of a supernatant comprising the clarifiedtest sample. In some embodiments of any of the aspects, a test samplecan be a pre-processed test sample, for example, supernatant or filtrateresulting from a treatment selected from the group consisting ofcentrifugation, filtration, thawing, purification, and any combinationsthereof In some embodiments of any of the aspects, the test sample canbe treated with a chemical and/or biological reagent. Chemical and/orbiological reagents can be employed to protect and/or maintain thestability of the sample, including biomolecules (e.g., nucleic acid andprotein) therein, during processing. One exemplary reagent is a proteaseinhibitor, which is generally used to protect or maintain the stabilityof protein during processing. The skilled artisan is well aware ofmethods and processes appropriate for pre-processing of biologicalsamples required for determination of the level of an expression productas described herein.

In some embodiments of any of the aspects, the methods, assays, andsystems described herein can further comprise a step of obtaining orhaving obtained a test sample from a subject. In some embodiments of anyof the aspects, the subject can be a human subject. In some embodimentsof any of the aspects, the subject can be a subject in need of treatmentfor (e.g. having or diagnosed as having) cancer or a subject at risk ofor at increased risk of developing cancer as described elsewhere herein.

In some embodiments of any of the aspects, the method further comprisesdetermining the expression level of at least one gene selected fromTSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),E-cadherin (CDH1), ZEB1, AHNAK, COMP, TSP5, BRD2, BRD3, miR103a, andSOX2-OT in tumor tissue obtained from the subject. In some embodimentsof any of the aspects, the method further comprises determining theexpression level of at least one gene selected from COMP, TSP5, BRD2,BRD3, miR103a, and SOX-2-OT in tumor tissue obtained from the subject.

As used herein, the term “cancer” relates generally to a class ofdiseases or conditions in which abnormal cells divide without controland can invade nearby tissues. Cancer cells can also spread to otherparts of the body through the blood and lymph systems. There are severalmain types of cancer. Carcinoma is a cancer that begins in the skin orin tissues that line or cover internal organs. Sarcoma is a cancer thatbegins in bone, cartilage, fat, muscle, blood vessels, or otherconnective or supportive tissue. Leukemia is a cancer that starts inblood-forming tissue such as the bone marrow, and causes large numbersof abnormal blood cells to be produced and enter the blood. Lymphoma andmultiple myeloma are cancers that begin in the cells of the immunesystem. Central nervous system cancers are cancers that begin in thetissues of the brain and spinal cord.

In some embodiments of any of the aspects, the cancer is a primarycancer. In some embodiments of any of the aspects, the cancer is amalignant cancer. As used herein, the term “malignant” refers to acancer in which a group of tumor cells display one or more ofuncontrolled growth (i.e., division beyond normal limits), invasion(i.e., intrusion on and destruction of adjacent tissues), and metastasis(i.e., spread to other locations in the body via lymph or blood). Asused herein, the term “metastasize” refers to the spread of cancer fromone part of the body to another. A tumor formed by cells that havespread is called a “metastatic tumor” or a “metastasis.” The metastatictumor contains cells that are like those in the original (primary)tumor. As used herein, the term “benign” or “non-malignant” refers totumors that may grow larger but do not spread to other parts of thebody. Benign tumors are self-limited and typically do not invade ormetastasize.

A “cancer cell” or “tumor cell” refers to an individual cell of acancerous growth or tissue. A tumor refers generally to a swelling orlesion formed by an abnormal growth of cells, which may be benign,pre-malignant, or malignant. Most cancer cells form tumors, but some,e.g., leukemia, do not necessarily form tumors. For those cancer cellsthat form tumors, the terms cancer (cell) and tumor (cell) are usedinterchangeably.

As used herein the term “neoplasm” refers to any new and abnormal growthof tissue, e.g., an abnormal mass of tissue, the growth of which exceedsand is uncoordinated with that of the normal tissues. Thus, a neoplasmcan be a benign neoplasm, premalignant neoplasm, or a malignantneoplasm.

A subject that has a cancer or a tumor is a subject having objectivelymeasurable cancer cells present in the subject's body. Included in thisdefinition are malignant, actively proliferative cancers, as well aspotentially dormant tumors or micrometastatses. Cancers which migratefrom their original location and seed other vital organs can eventuallylead to the death of the subject through the functional deterioration ofthe affected organs.

Examples of cancer include but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer;bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancerof the peritoneum; cervical cancer; choriocarcinoma; colon and rectumcancer; connective tissue cancer; cancer of the digestive system;endometrial cancer; esophageal cancer; eye cancer; cancer of the headand neck; gastric cancer (including gastrointestinal cancer);glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelialneoplasm.; kidney or renal cancer; larynx cancer; leukemia; livercancer; lung cancer (e.g., small-cell lung cancer, non-small cell lungcancer, adenocarcinoma of the lung, and squamous carcinoma of the lung);lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma;myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth,and pharynx); ovarian cancer; pancreatic cancer; prostate cancer;retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of therespiratory system; salivary gland carcinoma; sarcoma; skin cancer;squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer;uterine or endometrial cancer; cancer of the urinary system; vulvalcancer; as well as other carcinomas and sarcomas; as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome

A “cancer cell” is a cancerous, pre-cancerous, or transformed cell,either in vivo, ex vivo, or in tissue culture, that has spontaneous orinduced phenotypic changes that do not necessarily involve the uptake ofnew genetic material. Although transformation can arise from infectionwith a transforming virus and incorporation of new genomic nucleic acid,or uptake of exogenous nucleic acid, it can also arise spontaneously orfollowing exposure to a carcinogen, thereby mutating an endogenous gene.Transformation/cancer is associated with, e.g., morphological changes,immortalization of cells, aberrant growth control, foci formation,anchorage independence, malignancy, loss of contact inhibition anddensity limitation of growth, growth factor or serum independence, tumorspecific markers, invasiveness or metastasis, and tumor growth insuitable animal hosts such as nude mice.

In some embodiments of any of the aspects, the cancer is an epithelialcancer. In some embodiments of any of the aspects, the cancer is anepithelial adenocarcinoma. In some embodiments of any of the aspects,the cancer is esophageal cancer, pancreatic cancer, cervical cancer,colorectal cancer, gastric cancer, lung cancer, uterine caner, renalcancer, breast cancer, or prostate cancer. In some embodiments of any ofthe aspects, the cancer is breast and/or prostate cancer.

In some embodiments of any of the aspects, the subject is diabetic. Asused herein, “diabetes” refers to diabetes mellitus, a metabolic diseasecharacterized by a deficiency or absence of insulin secretion by thepancreas. As used throughout, “diabetes” includes Type 1, Type 2, Type3, and Type 4 diabetes mellitus unless otherwise specified herein. Theonset of diabetes is typically due to a combination of hereditary andenvironmental causes, resulting in abnormally high blood sugar levels(hyperglycemia). The two most common forms of diabetes are due to eithera diminished production of insulin (in type 1), or diminished responseby the body to insulin (in type 2 and gestational). Both lead tohyperglycemia, which largely causes the acute signs of diabetes:excessive urine production, resulting compensatory thirst and increasedfluid intake, blurred vision, unexplained weight loss, lethargy, andchanges in energy metabolism. Diabetes can cause many complications.Acute complications (hypoglycemia, ketoacidosis, or nonketotichyperosmolar coma) may occur if the disease is not adequatelycontrolled. Serious long-term complications (i.e. chronic side effects)include cardiovascular disease (doubled risk), chronic renal failure,retinal damage (which can lead to blindness), nerve damage (of severalkinds), and microvascular damage, which may cause impotence and poorwound healing. Poor healing of wounds, particularly of the feet, canlead to gangrene, and possibly to amputation. In some embodiments, thediabetes can be Type 2 diabetes. Type 2 diabetes (non-insulin-dependentdiabetes mellitus (NIDDM), or adult-onset diabetes) is a metabolicdisorder that is primarily characterized by insulin resistance(diminished response by the body to insulin), relative insulindeficiency, and hyperglycemia. In some embodiments, a subject can bepre-diabetic, which can be characterized, for example, as havingelevated fasting blood sugar or elevated post-prandial blood sugar.

Subjects having diabetes can be identified by a physician using currentmethods of diagnosing diabetes. Symptoms and/or complications ofdiabetes which characterize this condition and aid in diagnosis are wellknown in the art and include but are not limited to, weight loss, slowhealing, polyuria, polydipsia, polyphagiam headaches, itchy skin, andfatigue. Tests that may aid in a diagnosis of, e.g. diabetes include,but are not limited to, blood tests (e.g., for fasting glucose levels).A family history of diabetes, or exposure to risk factors for diabetes(e.g. overweight) can also aid in determining if a subject is likely tohave diabetes or in making a diagnosis of diabetes. In some embodimentsof any of the aspects, the subject is identified as diabetic when theyare determined to have HbA1c of 6.5% or greater, or by fasting glucoseor fasting insulin.

In some embodiments of any of the aspects, the subject is overweight. Insome embodiments of any of the aspects, the subject is obese.

The term “obesity” refers to excess fat in the body. Obesity can bedetermined by any measure accepted and utilized by those of skill in theart. Currently, an accepted measure of obesity is body mass index (BMI),which is a measure of body weight in kilograms relative to the square ofheight in meters. Generally, for an adult over age 20, a BMI betweenabout 18.5 and 24.9 is considered normal, a BMI between about 25.0 and29.9 is considered overweight, a BMI at or above about 30.0 isconsidered obese, and a BMI at or above about 40 is considered morbidlyobese. (See, e.g., Gallagher et al. (2000) Am J Clin Nutr 72:694-701.)These BMI ranges are based on the effect of body weight on increasedrisk for disease. Although BMI correlates with body fat, the relationbetween BMI and actual body fat differs with age and gender. Forexample, women are more likely to have a higher percent of body fat thanmen for the same BMI. Furthermore, the BMI threshold that separatesnormal, overweight, and obese can vary, e.g. with age, gender,ethnicity, fitness, and body type, amongst other factors. In someembodiments, a subject with obesity can be a subject with a body massindex of at least about 25 kg/m². In some embodiments, a subject withobesity can be a subject with a body mass index of at least about 30kg/m².

As described herein, the inventors have found that the expressionpatterns of certain genes in exosomes indicate whether the exosome willpromote or inhibit cancer progression. Accordingly, administration ofexosomes with gene expression patterns indicative of a cancer-inhibitingexosome phenotype can be administered to patients with a therapeuticeffect. In one aspect of any of the embodiments, described herein is amethod of treating cancer in a subject in need thereof, the methodcomprising administering to the subject exosomes which are:

-   -   a. from a non-diabetic and/or non-obese donor; and/or    -   b. determined to have an level of expression of at least one        gene selected from:        -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,            TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin            (VIM), E-cadherin (CDH1), ZEB1, and AHNAK; which is not            increased; and/or        -   a level of expression of at least one gene selected from:        -   miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and            miR320d; which is not increased, wherein the level of            expression is relative to the level of expression in a            exosome obtained from a healthy non-diabetic subject.            In one aspect of any of the embodiments, described herein is            a composition comprising exosomes which are:    -   a. from a non-diabetic and/or non-obese donor; and/or    -   b. determined to have an level of expression of at least one        gene selected from:        -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,            TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin            (VIM), E-cadherin (CDH1), ZEB1, and AHNAK; which is not            increased; and/or        -   a level of expression of at least one gene selected from:        -   miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and            miR320d; which is not increased, wherein the level of            expression is relative to the level of expression in a            exosome obtained from a healthy non-diabetic subject;            for use in a method of treating cancer in a subject in need            thereof.

As described herein, the inventors have discovered that misregulation ofcertain genes in exosomes drives cancer progression, particularly theepithelial-mesenchymal transition (EMT). Accordingly, administeringinhibitors of genes upregulated in cancer-driving exosomes, or agonistsof genes downregulated in cancer-driving exosomes can providetherapeutic effects. Accordingly, in one aspect of any of theembodiments, described herein is a method of treating cancer in asubject in need thereof, the method comprising administering to thesubject:

-   -   a. an inhibitor of at least one gene selected from:        -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,            TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin            (VIM), E-cadherin (CDH1), ZEB1, and AHNAK; which is not            increased; and/or    -   b. an agonist of at least one gene selected from:        -   miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and            miR320d.            In one aspect of any of the embodiments, described herein is            a composition or combination comprising:    -   a. an inhibitor of at least one gene selected from:        -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,            TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin            (VIM), E-cadherin (CDH1), ZEB1, and AHNAK; which is not            increased; and/or    -   b. an agonist of at least one gene selected from:        -   miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and            miR320d;            for use in a method of treating cancer in a subject in need            thereof.

In some embodiments of any of the aspects, the subject can beadministered (or the composition/combination can comprise) one or moreof the inhibitors or agonists, e.g., two or more inhibitors, two or moreagonists, or at least one inhibitor and at least one agonist.Combinations of reagents include those pair-wise combinations shown inthe following table:

Inhib- Inhib- Inhib- Inhib- Inhib- Inhib- Inhib- Inhib- Inhib- Inhib-Inhib- itor of itor of itor of itor of itor of itor of itor of itor ofitor of itor of itor of miR374a-5p miR-93-5p miR-28-3p miR-let-7b-3pmiR-375 TSP5 SNAI1 TWIST1 SNAI2 VIM CDH1 Inhibitor x x x x x x x x x xof miR374a-5p Inhibitor X x x x x x x x x x of miR-93-5p Inhibitor X X xx x x x x x x of miR-28-3p Inhibitor X X x x x x x x x x ofmiR-let-7b-3p Inhibitor X X x x x x x x x x of miR-375 Inhibitor X X x xx x x x x x of TSPS Inhibitor X X x x x x x x x x of SNAI1 Inhibitor X Xx x x x x x x x of TWIST1 Inhibitor X X x x x x x x x x of SNAI2Inhibitor X X x x x x x x x x of VIM Inhibitor X X x x x x x x x x ofCDH1 Inhibitor X X x x x x x x x x x of ZEB1 Inhibitor X X x x x x x x xx x of AHNAK Agonist of X X x x x x x x x x x miR424-5p Agonist X X x xx x x x x x x of miR-326 Agonist of X X x x x x x x x x x miR424-5pAgonist of X X x x x x x x x x x miR-27a-3p Agonist of X X x x x x x x xx x miR320b Agonist of X X x x x x x x x x x miR320d Inhib- Inhib-Agonist Agonist Agonist Agonist Agonist Agoinst itor of itor of of of ofof of of ZEB1 AHNAK miR424-5p miR-326 miR424-5p miR27a-3p miR320bmiR320d Inhibitor x x x x x x x X of miR374a-5p Inhibitor x x x x x x xX of miR-93-5p Inhibitor x x x x x x x x of miR-28-3p Inhibitor x x x xx x x x of miR-let-7b-3p Inhibitor x x x x x x x x of miR-375 Inhibitorx x x x x x x x of TSPS Inhibitor x x x x x x x x of SNAI1 Inhibitor x xx x x x x x of TWIST1 Inhibitor x x x x x x x x of SNAI2 Inhibitor x x xx x x x x of VIM Inhibitor x x x x x x x x of CDH1 Inhibitor x x x x x xx of ZEB1 Inhibitor x x x x x x x of AHNAK Agonist of x x x x x x xmiR424-5p Agonist x x x x x x x of miR-326 Agonist of x x x x x x xmiR424-5p Agonist of x x x x x x x miR-27a-3p Agonist of x x x x x x xmiR320b Agonist of x x x x x x x miR320d

As used herein, “inhibitor” refers to an agent which can decrease theexpression and/or activity of a target, e.g. by at least 10% or more,e.g. by 10% or more, 50% or more, 70% or more, 80% or more, 90% or more,95% or more, or 98% or more. The efficacy of an inhibitor of one or moretargets, e.g. its ability to decrease the level and/or activity of thetarget can be determined, e.g. by measuring the level of an expressionproduct of the target and/or the activity of the target. In someembodiments of any of the aspects, the inhibitor can be an inhibitorynucleic acid; an aptamer; an antibody reagent; an antibody; or a smallmolecule. An inhibitor of a target described herein can inhibit theactivity, expression, or accumulation of the target polypeptide.Inhibitors can include inhibitors that act directly on the target itself(e.g., that bind to the protein or transcript, e.g., direct inhibitors).

In some embodiments of any of the aspects, an inhibitor of a specifiedtarget is an antibody, antibody reagent, or antigen-binding fragmentthereof, that specifically binds to the target.

In some embodiments of any of the aspects, an inhibitor of a targetdescribed herein is an inhibitory nucleic acid. In some embodiments ofany of the aspects, inhibitors of the expression of a given gene can bean inhibitory nucleic acid. As used herein, “inhibitory nucleic acid”refers to a nucleic acid molecule which can inhibit the expression of atarget, e.g., double-stranded RNAs (dsRNAs), inhibitory RNAs (iRNAs),and the like. In some embodiments of any of the aspects, the inhibitorynucleic acid can be a silencing RNA (siRNA), microRNA (miRNA), or shorthairpin RNA (shRNA). Inhibitory nucleic acids can also include guidesequence molecules (e.g., a guide RNA) that function, e.g., incombination with an enzyme, to induce insertions, deletions, indels,and/or mutations of a target, thereby inhibiting the expression of thetarget.

In some embodiments of any of the aspects, an iNA comprises a sequencethat is complementary to at least a portion of a target sequencedescribed herein. In some embodiments of any of the aspects, an iNAcomprises a sequence at least 15 nucleotides in length that iscomplementary to at least a portion of a target sequence describedherein. In some embodiments of any of the aspects, an iNA comprises asequence at least 20 nucleotides in length that is complementary to atleast a portion of a target sequence described herein.

In some embodiments of any of the aspects, an iNA comprises a sequencethat is the reverse complement to at least a portion of a targetsequence described herein. In some embodiments of any of the aspects, aniNA comprises a sequence at least 15 nucleotides in length that is thereverse complement to at least a portion of a target sequence describedherein. In some embodiments of any of the aspects, an iNA comprises asequence at least 20 nucleotides in length that is the reversecomplement to at least a portion of a target sequence described herein.

In some embodiments of any of the aspects, an iNA comprises a sequencethat can specifically hybridize to at least a portion of a targetsequence described herein. In some embodiments of any of the aspects, aniNA comprises a sequence at least 15 nucleotides in length that canspecifically hybridize to at least a portion of a target sequencedescribed herein. In some embodiments of any of the aspects, an iNAcomprises a sequence at least 20 nucleotides in length that canspecifically hybridize to at least a portion of a target sequencedescribed herein.

Double-stranded RNA molecules (dsRNA) have been shown to block geneexpression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). The inhibitory nucleic acids described herein caninclude an RNA strand (the antisense strand) having a region which is 30nucleotides or less in length, i.e., 15-30 nucleotides in length,generally 19-24 nucleotides in length, which region is substantiallycomplementary to at least part the targeted mRNA transcript. The use ofthese iRNAs enables the targeted degradation of mRNA transcripts,resulting in decreased expression and/or activity of the target.

As used herein, the term “iRNA” refers to an agent that contains RNA (ormodified nucleic acids as described below herein) and which mediates thetargeted cleavage of an RNA transcript via an RNA-induced silencingcomplex (RISC) pathway. In some embodiments of any of the aspects, aniRNA as described herein effects inhibition of the expression and/oractivity of a target, e.g. a gene described herein. In some embodimentsof any of the aspects, contacting a cell with the inhibitor (e.g. aniRNA) results in a decrease in the target mRNA level in a cell by atleast about 5%, about 10%, about 20%, about 30%, about 40%, about 50%,about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up toand including 100% of the target mRNA level found in the cell withoutthe presence of the iRNA. In some embodiments of any of the aspects,administering an inhibitor (e.g. an iRNA) to a subject results in adecrease in the target mRNA level in the subject by at least about 5%,about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about70%, about 80%, about 90%, about 95%, about 99%, up to and including100% of the target mRNA level found in the subject without the presenceof the iRNA.

In some embodiments of any of the aspects, the iRNA can be a dsRNA. AdsRNA includes two RNA strands that are sufficiently complementary tohybridize to form a duplex structure under conditions in which the dsRNAwill be used. One strand of a dsRNA (the antisense strand) includes aregion of complementarity that is substantially complementary, andgenerally fully complementary, to a target sequence. The target sequencecan be derived from the sequence of an mRNA formed during the expressionof the target, e.g., it can span one or more intron boundaries. Theother strand (the sense strand) includes a region that is complementaryto the antisense strand, such that the two strands hybridize and form aduplex structure when combined under suitable conditions. Generally, theduplex structure is between 15 and 30 base pairs in length inclusive,more generally between 18 and 25 base pairs in length inclusive, yetmore generally between 19 and 24 base pairs in length inclusive, andmost generally between 19 and 21 base pairs in length, inclusive.Similarly, the region of complementarity to the target sequence isbetween 15 and 30 base pairs in length inclusive, more generally between18 and 25 base pairs in length inclusive, yet more generally between 19and 24 base pairs in length inclusive, and most generally between 19 and21 base pairs in length nucleotides in length, inclusive. In someembodiments of any of the aspects, the dsRNA is between 15 and 20nucleotides in length, inclusive, and in other embodiments, the dsRNA isbetween 25 and 30 nucleotides in length, inclusive. As the ordinarilyskilled person will recognize, the targeted region of an RNA targetedfor cleavage will most often be part of a larger RNA molecule, often anmRNA molecule. Where relevant, a “part” of an mRNA target is acontiguous sequence of an mRNA target of sufficient length to be asubstrate for RNAi-directed cleavage (i.e., cleavage through a RISCpathway). dsRNAs having duplexes as short as 9 base pairs can, undersome circumstances, mediate RNAi-directed RNA cleavage. Most often atarget will be at least 15 nucleotides in length, preferably 15-30nucleotides in length.

Exemplary embodiments of types of inhibitory nucleic acids can include,e.g,. siRNA, shRNA, miRNA, and/or amiRNA, which are well known in theart. One skilled in the art would be able to design further siRNA,shRNA, or miRNA to target the nucleic acid sequence of a targetdescribed herein, e.g., using publically available design tools. siRNA,shRNA, or miRNA is commonly made using companies such as Dharmacon(Layfayette, Colo.) or Sigma Aldrich (St. Louis, Mo.).

In some embodiments of the various aspects described herein, theinhibitory nucleic acid is a guide nucleic acid (gNA). As used herein,the terms “guide nucleic acid,” “guide sequence,” “crRNA,” “guide RNA,”“single guide RNA,” “gRNA” or “CRISPR guide sequence” refer to a nucleicacid comprising a sequence that determines the specificity of an enzyme,e.g., the Cas DNA binding protein of a CRISPR/Cas system, to apolynucleotide target. The gNA can comprise a polynucleotide sequencewith at least partial complementarity with a target nucleic acidsequence, sufficient to hybridize with the target nucleic acid sequenceand to direct sequence-specific binding of an enzyme, e.g, a nuclease,to the target nucleic acid sequence.

In some embodiments, the enzyme directed by the gNA is a gene-editingprotein, e.g., any nuclease that induces a nick or double-strand breakinto a desired recognition site. Such enzymes can be native orengineered. These breaks can then be repaired by the cell in one of twoways: non-homologous end joining and homology-directed repair(homologous recombination). In non-homologous end joining (NHEJ), thedouble-strand breaks are repaired by direct ligation of the break endsto one another. As such, no new nucleic acid material is inserted intothe site, although some nucleic acid material may be lost, resulting ina deletion. In homology-directed repair, a donor polynucleotide withhomology to the cleaved target DNA sequence can be used as a templatefor repair of the cleaved target DNA sequence, resulting in the transferof genetic information from the donor polynucleotide to the target DNA.Therefore, new nucleic acid material may be inserted/copied into thesite. The modifications of the target DNA due to NHEJ and/orhomology-directed repair can be used for gene correction, genereplacement, gene tagging, transgene insertion, nucleotide deletion,gene disruption, gene mutation, etc.

In one embodiment, the gene-editing protein is a CRISPR-associatednuclease. The native prokaryotic CRISPR-associated nuclease systemcomprises an array of short repeats with intervening variable sequencesof constant length (i.e., clusters of regularly interspaced shortpalindromic repeats), and CRISPR-associated (“Cas”) nuclease proteins.The RNA of the transcribed CRISPR array is processed by a subset of theCas proteins into small guide RNAs, which generally have two componentsas discussed below. There are at least three different systems: Type I,Type II and Type III. The enzymes involved in the processing of the RNAinto mature crRNA are different in the 3 systems. In the nativeprokaryotic system, the guide RNA (“gRNA”) comprises two short,non-coding RNA species referred to as CRISPR RNA (“crRNA”) andtrans-acting RNA (“tracrRNA”). In an exemplary system, the gRNA forms acomplex with a nuclease, for example, a Cas nuclease. The gRNA: nucleasecomplex binds a target polynucleotide sequence having a protospaceradjacent motif (“PAM”) and a protospacer, which is a sequencecomplementary to a portion of the gRNA. The recognition and binding ofthe target polynucleotide by the gRNA: nuclease complex induces cleavageof the target.

Any CRISPR-associated nuclease can be used in the system and methods ofthe invention. CRISPR nuclease systems are known to those of skill inthe art, e.g. Cas9, Cas12, Cas12a, or the like, see U.S Pat. No.8,993,233, US 2015/0291965, US 2016/0175462, US 2015/0020223, US2014/0179770, U.S Pat. Nos. 8,697,359; 8,771,945; 8, 795,965; WO2015/191693; U.S. Pat. No. 8,889,418; WO 2015/089351; WO 2015/089486; WO2016/028682; WO 2016/049258; WO 2016/094867; WO 2016/094872; WO2016/094874; WO 2016/112242; US 2016/0153004; US 2015/0056705; US2016/0090607; US 2016/0029604; U.S Pat. Nos. 8,865,406; 8,871,445; eachof which are incorporated by reference in their entirety. The nucleasecan also be a phage Cas nuclease, e.g., CasΦ (e.g., Pausch et al.Science 369:333-7 (2020); which is incorporated by reference herein inits entirety).

The full-length guide nucleic acid strand can be any length. Forexample, the guide nucleic acid strand can be about or more than about5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. Insome embodiments of the various aspects described herein, a nucleic acidstrand is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, orfewer nucleotides in length. For example, the guide nucleic acidsequence is 10-30 nucleotides long.

In addition to a sequence that is complementary to a target nucleicacid, in some embodiments, the gNA also comprises a scaffold sequence.Expression of a gNA encoding both a sequence complementary to a targetnucleic acid and scaffold sequence has the dual function of both binding(hybridizing) to the target nucleic acid and recruiting the endonucleaseto the target nucleic acid, which may result in site-specific CRISPRactivity. In some embodiments, such a chimeric gNA may be referred to asa single guide RNA (sgRNA).

In some embodiments of the various aspects described herein, the guidenucleic acid is designed using a guide design tool (e.g., Benchling™;Broad Institute GPP™; CasOFFinder™; CHOPCHOP™; CRISPOR™; Deskgen™;E-CRISP™; Geneious™; GenHub™; GUIDES™ (e.g., for library design);Horizon Discovery™; IDT™; Off-Spotter™; and Synthego™; which areavailable on the world wide web).

In some embodiments of any of the aspects, the RNA of an iRNA, e.g., adsRNA, is chemically modified to enhance stability or other beneficialcharacteristics. The nucleic acids described herein may be synthesizedand/or modified by methods well established in the art, such as thosedescribed in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, whichis hereby incorporated herein by reference. Modifications include, forexample, (a) end modifications, e.g., 5′ end modifications(phosphorylation, conjugation, inverted linkages, etc.) 3′ endmodifications (conjugation, DNA nucleotides, inverted linkages, etc.),(b) base modifications, e.g., replacement with stabilizing bases,destabilizing bases, or bases that base pair with an expanded repertoireof partners, removal of bases (abasic nucleotides), or conjugated bases,(c) sugar modifications (e.g., at the 2′ position or 4′ position) orreplacement of the sugar, as well as (d) backbone modifications,including modification or replacement of the phosphodiester linkages.Specific examples of RNA compounds useful in the embodiments describedherein include, but are not limited to RNAs containing modifiedbackbones or no natural internucleoside linkages. RNAs having modifiedbackbones include, among others, those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified RNAs that do not have aphosphorus atom in their internucleoside backbone can also be consideredto be oligonucleosides. In some embodiments of any of the aspects, themodified RNA will have a phosphorus atom in its internucleosidebackbone.

Modified RNA backbones can include, for example, phosphorothioates,chiral phosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included. Modified RNAbackbones that do not include a phosphorus atom therein have backbonesthat are formed by short chain alkyl or cycloalkyl internucleosidelinkages, mixed heteroatoms and alkyl or cycloalkyl internucleosidelinkages, or one or more short chain heteroatomic or heterocyclicinternucleoside linkages. These include those having morpholino linkages(formed in part from the sugar portion of a nucleoside); siloxanebackbones; sulfide, sulfoxide and sulfone backbones; formacetyl andthioformacetyl backbones; methylene formacetyl and thioformacetylbackbones; alkene containing backbones; sulfamate backbones;methyleneimino and methylenehydrazino backbones; sulfonate andsulfonamide backbones; amide backbones; others having mixed N, 0, S andCH2 component parts, and oligonucleosides with heteroatom backbones, andin particular —CH2—NH—CH2—, —CH2-N(CH3)-O—CH2- [known as a methylene(methylimino) or MMI backbone], —CH2-O—N(CH3)-CH2-,—CH2-N(CH3)-N(CH3)-CH2- and —N(CH3)-CH2-CH2- [wherein the nativephosphodiester backbone is represented as —O—P—O—CH2-].

In other RNA mimetics suitable or contemplated for use in iRNAs, boththe sugar and the internucleoside linkage, i.e., the backbone, of thenucleotide units are replaced with novel groups. The base units aremaintained for hybridization with an appropriate nucleic acid targetcompound. One such oligomeric compound, an RNA mimetic that has beenshown to have excellent hybridization properties, is referred to as apeptide nucleic acid (PNA). In PNA compounds, the sugar backbone of anRNA is replaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone.

The RNA of an iRNA can also be modified to include one or more lockednucleic acids (LNA). A locked nucleic acid is a nucleotide having amodified ribose moiety in which the ribose moiety comprises an extrabridge connecting the 2′ and 4′ carbons. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects (Elmen, J. et al.,(2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic AcidsResearch 31(12):3185-3193).

Modified RNAs can also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, described herein can include one of thefollowing at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S—or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkylor C2 to C10 alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH2)nO] mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2) nCH3,O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 toabout 10. In some embodiments of any of the aspects, dsRNAs include oneof the following at the 2′ position: C1 to C10 lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN,Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments of any of the aspects, the modification includes a 2′methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or2′-MOE) (Martin et al., Hely. Chim. Acta, 1995, 78:486-504) i.e., analkoxy-alkoxy group. Another exemplary modification is2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as2′-DMAOE, as described in examples herein below, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH2-O—CH2-N(CH2)2, also described in examples herein below.

Other modifications include 2′-methoxy (2′-OCH3), 2′-aminopropoxy(2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs may alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar.

An inhibitory nucleic acid can also include nucleobase (often referredto in the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo,particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and3-deazaadenine. Certain of these nucleobases are particularly useful forincreasing the binding affinity of the inhibitory nucleic acids featuredin the invention. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., Eds., dsRNA Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are exemplary base substitutions, evenmore particularly when combined with 2′-O-methoxyethyl sugarmodifications.

The preparation of the modified nucleic acids, backbones, andnucleobases described above are well known in the art.

Another modification of an inhibitory nucleic acid featured in theinvention involves chemically linking to the inhibitory nucleic acid toone or more ligands, moieties or conjugates that enhance the activity,cellular distribution, pharmacokinetic properties, or cellular uptake ofthe iRNA. Such moieties include but are not limited to lipid moietiessuch as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci.USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med.Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan etal., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990,259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), aphospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res.,1990, 18:3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In some embodiments of any of the aspects, an inhibitor of a targetdescribed herein can comprise an antibody reagent. Antibody reagentsspecific for the targets and/or markers described herein, are known inthe art. For example, such reagents are readily commercially availableas shown in Table 1 below.

TABLE 1 Exemplary antibodies Name Company Catalog Number Human COMPmonoclonal ProteinTech 66793-1-Ig antibody Anti-COMP Millipore SigmaMABT36 (Thrombospondin-5) Antibody, clone 484D1 Anti-Thrombospondin 5Santa Cruz sc-374660 Antibody (F-7) Biotechnologies Anti-Thrombospondin5 Santa Cruz sc-59941 Antibody (MA37C94) BiotechnologiesAnti-Thrombospondin 5 Santa Cruz sc-33696 Antibody (644A8D5)Biotechnologies Human R&D Systems MAB3134 COMP/Thrombospondin-5 AntibodyMonoclonal Anti-SNAI1 Millipore Sigma SAB1404386- antibody produced in100UG mouse Anti-SNAI1 Antibody, Millipore Sigma MABE167 clone 10H4.1Monoclonal Anti-SNAI1 Millipore Sigma AMAB91215- antibody produced in25UL mouse (clone CL3700) SNAIL Monoclonal Invitrogen MA5-14801 Antibody(F.31.8) SNAI1/Snail antibody Active Motif Catalog No: (mAb) Clone:9H261367 Anti-SNAI1 monoclonal Creative DCABH-2356 antibody (clone 7E3)Diagnostics Anti-SNAI1 monoclonal Creative DCABH-6168 antibody (To0I3)Diagnostics Anti-Human SNAIL Creative CABT-L1601 monoclonal antibody(clone Diagnostics G.42.9) Anti-SNAI 1 Antibody (G- Santa Cruz sc-2719777) Biotechnologies Anti-SNAI 1 Antibody (E- Santa Cruz sc-393172 10)Biotechnologies TWIST1 (E7E2G) Rabbit Cell Signaling #69366 mAbTechnology TWIST1 Monoclonal Invitrogen MA5-17195 Antibody (2F8E7)TWIST1 Monoclonal Invitrogen MA5-38652 Antibody (10E4E6) TWIST1Monoclonal Invitrogen MA5-32927 Antibody (3E1) TWIST1 Monoclonal AbnovaH00007291- Antibody (2F8) M03 Twist-1 Antibody Novus H00007291- (3A2)Biologicals M04 Anti-twist Antibody Santa Cruz sc-81417 (Twist2C1a)Biotechnologies Anti-SLUG Antibody Santa Cruz sc-166476 (A-7)Biotechnologies Slug (C19G7) Rabbit Cell Signaling #9585 mAb TechnologyPE Mouse anti-SNAI2/ BD Biosciences 564615 Slug (Clone S43-1259) SLUGMonoclonal Invitrogen MA5-26385 Antibody (OTI1A6) SNAI2 MonoclonalOrigene TA800196 Antibody (OTI1G7) SLUG Monoclonal Invitrogen MA5-38634Antibody (4B6D5) SNAI2 Monoclonal Abnova H00006591- Antibody (3C12) M05Anti-SNAI2 monoclonal Creative DCABH-2021 antibody (2H8) DiagnosticsAnti-Vimentin Antibody Santa Cruz sc-373717 (E-5) BiotechnologiesVimentin Antibody Novus Biologicals MAB2105 (280618) Vimentin Antibody(2D1) Novus Biologicals NBP1-92687 Vimentin Monoclonal Progen61013PROGEN Antibody (VIM 3B4) Vimentin Antibody Novus BiologicalsNBP3-08936 (VIM/1937R) Anti-Vimentin Antibody Santa Cruz sc-6260 (V9)Biotechnologies Vimentin Monoclonal Invitrogen MA3-745 Antibody (J144)Vimentin Monoclonal Invitrogen OMA1-06001 Antibody (RV202) VIMMonoclonal Antibody Origene TA801297 (OTI1A9), TrueMAB ™ Anti-E-cadherinAntibody Santa Cruz sc-21791 (67A4) Biotechnologies Anti-E Cadherinantibody Abcam ab219332 [CDH1/1525] E-cadherin Monoclonal Invitrogen #13-5700 Antibody (SHE78-7) E-cadherin Monoclonal Invitrogen # 13-1700Antibody (HECD-1) CDH1 Monoclonal Origene # UM800076 Antibody (UMAB184),UltraMAB ™ E-Cadherin Monoclonal AbboMax # 605-730 Antibody (4A2C7)E-cadherin Monoclonal Proteintech # 60335-1-IG Antibody (6B11F11)Anti-ZEB1 Antibody Santa Cruz sc-515797 (H-3) Biotechnologies ZEB1Monoclonal Invitrogen # 14-9741-82 Antibody (3G6), eBioscience ™ ZEB1Monoclonal Origene # TA802298 Antibody (OTI3G6), TrueMAB ™ ZEB1Monoclonal Proteintech # 66279-1-IG Antibody (1H1F1) ZEB1 RecombinantRabbit Bethyl # A700-102 Monoclonal Antibody Laboratories (BLR102H) ZEB1Monoclonal OriGene # CF802313 Antibody (OTI7E12), TrueMAB ™ Anti-AHNAKAntibody Santa Cruz sc-390743 (E-5) Biotechnologies Anti-AHNAK AntibodySanta Cruz sc-134252 (1G11) Biotechnologies AHNAK Monoclonal Invitrogen# MA1-10050 Antibody (EM-09) AHNAK Monoclonal Abnova # H00079026-Antibody (3G7) M01 Anti-AHNAK monoclonal Creative DCABH-9479 antibody(clone FN-10) Diagnostics AHNAK polyclonal Abnova # H00079026- antibody(A01) A01 AHNAK Antibody (clone LifeSpan LS-C342403 2014C3a) LS-C342403Biosciences

In some embodiments of any of the aspects, an antibody reagent specificfor a target and/or maker described herein (e.g., that bindsspecifically to and inhibits the target and/or marker) can be anantibody reagent comprising one or more (e.g., one, two, three, four,five, or six) CDRs of any one of the antibodies recited in Table 1. Insome embodiments of any of the aspects, an antibody reagent specific fora target and/or maker described herein (e.g., that binds specifically toand inhibits the target and/or marker) can be an antibody reagentcomprising the six CDRs of any one of the antibodies recited in Table 1.In some embodiments of any of the aspects, an antibody reagent specificfor a target and/or maker described herein (e.g., that bindsspecifically to and inhibits the target and/or marker) can be anantibody reagent comprising the three heavy chain CDRs of any one of theantibodies recited in Table 1. In some embodiments of any of theaspects, an antibody reagent specific for a target and/or makerdescribed herein (e.g., that binds specifically to and inhibits thetarget and/or marker) can be an antibody reagent comprising the threelight chain CDRs of any one of the antibodies recited in Table 1. Insome embodiments of any of the aspects, an antibody reagent specific fora target and/or maker described herein (e.g., that binds specifically toand inhibits the target and/or marker) can be an antibody reagentcomprising the VH and/or VL domains of any one of the antibodies recitedin Table 1. In some embodiments of any of the aspects, an antibodyreagent specific for a target and/or maker described herein (e.g., thatbinds specifically to and inhibits the target and/or marker) can be anantibody reagent comprising the VH and VL domains of any one of theantibodies recited in Table 1. Such antibody reagents are specificallycontemplated for use in the methods and/or compositions describedherein.

As used herein, the term “agonist” refers to an agent which increasesthe expression and/or activity of the target by at least 10% or more,e.g. by 10% or more, 50% or more, 100% or more, 200% or more, 500% ormore, or 1000% or more. The efficacy of an agonist, e.g. its ability toincrease the level and/or activity of the target can be determined, e.g.by measuring the level of an expression product of the target and/or theactivity of the target. Methods for measuring the level of a given mRNAand/or polypeptide are known to one of skill in the art, e.g. RTPCR withprimers can be used to determine the level of RNA, and Western blottingwith an antibody can be used to determine the level of a polypeptide.Suitable primers for a given target are readily identified by one ofskill in the art, e.g., using software widely available for this purpose(e.g., Primer3 or PrimerBank, which are both available on the world wideweb). Antibodies to polypeptide gene expression products of the immuneresponse regulators described herein are commercially available, e.g.,from AbCam (Cambridge, Mass.). Assays for measuring the activity of thetargets described herein are provided elsewhere herein. In someembodiments of any of the aspects, an agonist of a given polypeptide canbe the polypeptide, a nucleic acid encoding the polypeptide, or a smallmolecule.

Non-limiting examples of agonists of a given polypeptide target, caninclude the target polypeptides or variants or functional fragmentsthereof and nucleic acids encoding the polypeptide or variants orfunctional fragments thereof. In some embodiments of any of the aspects,the agonist of a given target, is a polypeptide of that target orvariants or functional fragment thereof and/or a nucleic acid encodingthe polypeptide or variant or functional fragment thereof. In someembodiments of any of the aspects, the polypeptide agonist can be anengineered and/or recombinant polypeptide. In some embodiments of any ofthe aspects, the polypeptide agonist can be a nucleic acid encoding apolypeptide, e.g. a functional fragment thereof In some embodiments ofany of the aspects, the nucleic acid can be comprised by a vector.

In some embodiments of any of the aspects, a polypeptide agonist cancomprise one of the sequences described herein for each target. In someembodiments of any of the aspects, a polypeptide agonist can consistessentially of one of the sequences provided below herein for eachtarget. In some embodiments of any of the aspects, a polypeptide agonistcan consist of one of the sequences provided below herein for eachtarget. In some embodiments of any of the aspects, an agonist cancomprise a nucleic acid encoding one of the sequences provided belowherein for each target. In some embodiments of any of the aspects, anagonist can be a polypeptide comprising a reference/wild-type sequencedescribed herein with at least 80%, at least 85%, at least 90%, at least95%, or at least 98% identity to the reference/wild-type sequence andwhich retains the activity of the reference/wild-type sequence. In someembodiments of any of the aspects, an agonist can be a polypeptidecomprising a reference/wild-type sequence described herein with at least95% identity to the reference/wild-type sequence and which retains theactivity of the reference/wild-type sequence.

In some embodiments of any of the aspects, the agonist is an exogenouspolypeptide. In some embodiments of any of the aspects, the subject isadministered exogenous polypeptide, e.g., the polypeptide is produced invitro and/or synthesized and purified polypeptide is provided to thesubject. In some embodiments of any of the aspects, the agonist is anectopic polypeptide. In some embodiments of any of the aspects, thesubject is administered ectopic polypeptide, e.g., the polypeptide isproduced in vitro and/or synthesized and purified polypeptide isprovided to the subject.

In some embodiments of any of the aspects, the agonist can be a nucleicacid encoding a polypeptide (or a variant or functional fragmentthereof) and/or a vector comprising a nucleic acid encoding apolypeptide (or a variant or functional fragment thereof). A nucleicacid encoding a polypeptide can be, e.g., an RNA molecule, a plasmid,and/or an expression vector. In some embodiments of any of the aspects,the nucleic acid encoding a polypeptide can be an mRNA. In someembodiments of any of the aspects, the nucleic acid encoding apolypeptide can be a modified mRNA. In some embodiments of any of theaspects, the agonist can be a nucleic acid encoding a polypeptide, e.g.,exogenous and/or ectopic polypeptide. In some embodiments of any of theaspects, the subject is administered the nucleic acid encoding exogenousand/or ectopic polypeptide, e.g., the nucleic acid is transcribed and/ortranslated after the administering step to provide exogenous and/orectopic polypeptide to the subject.

In some embodiments of any of the aspects, a polypeptide or nucleic acidas described herein can be engineered. As used herein, “engineered”refers to the aspect of having been manipulated by the hand of man. Forexample, a polypeptide is considered to be “engineered” when at leastone aspect of the polypeptide, e.g., its sequence, has been manipulatedby the hand of man to differ from the aspect as it exists in nature. Asis common practice and is understood by those in the art, progeny of anengineered cell are typically still referred to as “engineered” eventhough the actual manipulation was performed on a prior entity.

In some embodiments of any of the aspects, the agonist and/or inhibitoris administered as a nucleic acid. In some embodiments of any of theaspects, a nucleic acid encoding the agonist and/or inhibitor isadministered. In some embodiments of any of the aspects, the subject isadministered a vector comprising a nucleic acid. Vectors can be, e.g., aDNA or RNA vector.

In some embodiments of any of the aspects, the methods described hereinrelate to treating a subject having or diagnosed as having cancer with acomposition or combination as described herein. Subjects having cancercan be identified by a physician using current methods of diagnosingcancer. Symptoms and/or complications of cancer which characterize theseconditions and aid in diagnosis are well known in the art and includebut are not limited to, for example, a lump/mass/tumor, swelling, orpain. Tests that may aid in a diagnosis of, e.g. cancer include, but arenot limited to, x-rays, MRI, ultrasound, a biopsy, or tests for thefunction/activity of affected organs or systems. A family history ofcancer or exposure to risk factors for cancer (e.g. smoke, radiation,pollutants, mutation, etc.) can increase the risk of a subject havingcancer.

In some embodiments of any of the aspects, the subject is one determinedto have an increased level of expression of at least one gene selectedfrom:

-   -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5,        Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),        E-cadherin (CDH1), ZEB1, and AHNAK; or        a decreased level of expression of at least one gene selected        from:    -   miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d.

In some embodiments of any of the aspects, the method further comprisesa first step of determining, in the subject or a sample obtained fromthe subject, the level of expression of at least one gene selected from:

-   -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5,        Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),        E-cadherin (CDH1), ZEB1, and AHNAK; miR424-5p, miR-326,        miR424-5p, miR-27a-3p, miR320b and miR320d.        In some embodiments of any of the aspects, the method further        comprises a first step of determining that the subject has an        increased level of expression of at least one gene selected        from:    -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5,        Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),        E-cadherin (CDH1), ZEB1, and AHNAK; or        a decreased level of expression of at least one gene selected        from:    -   miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d.

The compositions and methods described herein can be administered to asubject having or diagnosed as having cancer. In some embodiments of anyof the aspects, the methods described herein comprise administering aneffective amount of compositions described herein to a subject in orderto alleviate a symptom of a cancer. As used herein, “alleviating asymptom” of a cancer is ameliorating any condition or symptom associatedwith the cancer. As compared with an equivalent untreated control, suchreduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99%or more as measured by any standard technique. A variety of means foradministering the compositions described herein to subjects are known tothose of skill in the art. Such methods can include, but are not limitedto oral, parenteral, intravenous, intramuscular, subcutaneous,transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection,or intratumoral administration. Administration can be local or systemic.In some embodiments of any of the apsects, the administration issubcutaneous.

In some embodiments of any of the aspects, the subject in need oftreatment for cancer, or the subject administered a composition orcombination as described herein is diabetic. In some embodiments of anyof the aspects, the subject in need of treatment for cancer, or thesubject administered a composition or combination as described herein isobese. In some embodiments of any of the aspects, the subject in need oftreatment for cancer, or the subject administered a composition orcombination as described herein is diabetic and obese.

In some embodiments of any of the aspects, the methods of treatmentdescribed herein reduce EMT in the subject. EMT is a process by whichepithelial cells lose their cell polarity and cell-cell adhesion, andgain migratory and invasive properties to become mesenchymal stem cells.EMT can be detected by measuring the expression of EMT marker genes,e.g, increases in N-cadherin, fibronectin, and/or vimentin and/or adecrease in E-cadherin in cancer cells are markers of EMT. In someembodiments of any of the aspects, the methods of treatment describedherein reduce metastasis in the subject.

The term “effective amount” as used herein refers to the amount of acomposition or combination needed to alleviate at least one or moresymptom of the disease or disorder, and relates to a sufficient amountof pharmacological composition to provide the desired effect. The term“therapeutically effective amount” therefore refers to an amount of anagent or composition that is sufficient to provide a particularanti-cancer effect when administered to a typical subject, e,g., adecrease in tumor size, tumor growth, or EMT prevelance or rate. Aneffective amount as used herein, in various contexts, would also includean amount sufficient to delay the development of a symptom of thedisease, alter the course of a symptom disease (for example but notlimited to, slowing the progression of a symptom of the disease), orreverse a symptom of the disease. Thus, it is not generally practicableto specify an exact “effective amount”. However, for any given case, anappropriate “effective amount” can be determined by one of ordinaryskill in the art using only routine experimentation.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dosage can vary depending upon the dosage formemployed and the route of administration utilized. The dose ratiobetween toxic and therapeutic effects is the therapeutic index and canbe expressed as the ratio LD50/ED50. Compositions and methods thatexhibit large therapeutic indices are preferred. A therapeuticallyeffective dose can be estimated initially from cell culture assays.Also, a dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC50 (i.e., theconcentration of the active ingredient, which achieves a half-maximalinhibition of symptoms) as determined in cell culture, or in anappropriate animal model. Levels in plasma can be measured, for example,by high performance liquid chromatography. The effects of any particulardosage can be monitored by a suitable bioassay, e.g., assay for EMT,among others. The dosage can be determined by a physician and adjusted,as necessary, to suit observed effects of the treatment.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the minimal effective dose and/or maximaltolerated dose. The dosage can vary depending upon the dosage formemployed and the route of administration utilized. A therapeuticallyeffective dose can be estimated initially from cell culture assays.Also, a dose can be formulated in animal models to achieve a dosagerange between the minimal effective dose and the maximal tolerated dose.The effects of any particular dosage can be monitored by a suitablebioassay, e.g., assay for tumor growth and/or size among others. Thedosage can be determined by a physician and adjusted, as necessary, tosuit observed effects of the treatment.

In some embodiments, the technology described herein relates to apharmaceutical composition comprising an exosome, inhibitor, agonist,and/or other reagents as described herein, and optionally apharmaceutically acceptable carrier. In some embodiments, the activeingredients of the pharmaceutical composition comprise an exosome,inhibitor, agonist, and/or other reagents as described herein. In someembodiments, the active ingredients of the pharmaceutical compositionconsist essentially of an exosome, inhibitor, agonist, and/or otherreagents as described herein. In some embodiments, the activeingredients of the pharmaceutical composition consist of an exosome,inhibitor, agonist, and/or other reagents as described herein.Pharmaceutically acceptable carriers and diluents include saline,aqueous buffer solutions, solvents and/or dispersion media. The use ofsuch carriers and diluents is well known in the art. Some non-limitingexamples of materials which can serve as pharmaceutically-acceptablecarriers include: (1) sugars, such as lactose, glucose and sucrose; (2)starches, such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, methylcellulose,ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, suchas magnesium stearate, sodium lauryl sulfate and talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C₂-C₁₂ alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” or the like are used interchangeably herein. In someembodiments, the carrier inhibits the degradation of the active agent,e.g. an exosome, inhibitor, agonist, and/or other reagents as describedherein.

In some embodiments, the pharmaceutical composition comprising anexosome, inhibitor, agonist, and/or other reagents as described hereincan be a parenteral dose form. Since administration of parenteral dosageforms typically bypasses the patient's natural defenses againstcontaminants, parenteral dosage forms are preferably sterile or capableof being sterilized prior to administration to a patient. Examples ofparenteral dosage forms include, but are not limited to, solutions readyfor injection, dry products ready to be dissolved or suspended in apharmaceutically acceptable vehicle for injection, suspensions ready forinjection, and emulsions. In addition, controlled-release parenteraldosage forms can be prepared for administration of a patient, including,but not limited to, DUROS®-type dosage forms and dose-dumping.

Suitable vehicles that can be used to provide parenteral dosage forms ofa composition or combination as disclosed within are well known to thoseskilled in the art. Examples include, without limitation: sterile water;water for injection USP; saline solution; glucose solution; aqueousvehicles such as but not limited to, sodium chloride injection, Ringer'sinjection, dextrose Injection, dextrose and sodium chloride injection,and lactated Ringer's injection; water-miscible vehicles such as, butnot limited to, ethyl alcohol, polyethylene glycol, and propyleneglycol; and non-aqueous vehicles such as, but not limited to, corn oil,cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropylmyristate, and benzyl benzoate. Compounds that alter or modify thesolubility of a pharmaceutically acceptable salt of an active ingredientas disclosed herein can also be incorporated into the parenteral dosageforms of the disclosure, including conventional and controlled-releaseparenteral dosage forms.

Pharmaceutical compositions comprising an exosome, inhibitor, agonist,and/or other reagents can also be formulated to be suitable for oraladministration, for example as discrete dosage forms, such as, but notlimited to, tablets (including without limitation scored or coatedtablets), pills, caplets, capsules, chewable tablets, powder packets,cachets, troches, wafers, aerosol sprays, or liquids, such as but notlimited to, syrups, elixirs, solutions or suspensions in an aqueousliquid, a non-aqueous liquid, an oil-in-water emulsion, or awater-in-oil emulsion. Such compositions contain a predetermined amountof the pharmaceutically acceptable salt of the disclosed compounds, andmay be prepared by methods of pharmacy well known to those skilled inthe art. See generally, Remington: The Science and Practice of Pharmacy,21st Ed., Lippincott, Williams, and Wilkins, Philadelphia Pa. (2005).

Conventional dosage forms generally provide rapid or immediate drugrelease from the formulation. Depending on the pharmacology andpharmacokinetics of the drug, use of conventional dosage forms can leadto wide fluctuations in the concentrations of the drug in a patient'sblood and other tissues. These fluctuations can impact a number ofparameters, such as dose frequency, onset of action, duration ofefficacy, maintenance of therapeutic blood levels, toxicity, sideeffects, and the like. Advantageously, controlled-release formulationscan be used to control a drug's onset of action, duration of action,plasma levels within the therapeutic window, and peak blood levels. Inparticular, controlled- or extended-release dosage forms or formulationscan be used to ensure that the maximum effectiveness of a drug isachieved while minimizing potential adverse effects and safety concerns,which can occur both from under-dosing a drug (i.e., going below theminimum therapeutic levels) as well as exceeding the toxicity level forthe drug. In some embodiments, the composition or combination can beadministered in a sustained release formulation.

Controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledrelease counterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include: 1) extended activity of the drug; 2) reduceddosage frequency; 3) increased patient compliance; 4) usage of lesstotal drug; 5) reduction in local or systemic side effects; 6)minimization of drug accumulation; 7) reduction in blood levelfluctuations; 8) improvement in efficacy of treatment; 9) reduction ofpotentiation or loss of drug activity; and 10) improvement in speed ofcontrol of diseases or conditions. Kim, Cherng-ju, Controlled ReleaseDosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release other amountsof drug to maintain this level of therapeutic or prophylactic effectover an extended period of time. In order to maintain this constantlevel of drug in the body, the drug must be released from the dosageform at a rate that will replace the amount of drug being metabolizedand excreted from the body. Controlled-release of an active ingredientcan be stimulated by various conditions including, but not limited to,pH, ionic strength, osmotic pressure, temperature, enzymes, water, andother physiological conditions or compounds.

A variety of known controlled- or extended-release dosage forms,formulations, and devices can be adapted for use with the salts andcompositions of the disclosure. Examples include, but are not limitedto, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809;3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548;5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1 ; each ofwhich is incorporated herein by reference. These dosage forms can beused to provide slow or controlled-release of one or more activeingredients using, for example, hydroxypropylmethyl cellulose, otherpolymer matrices, gels, permeable membranes, osmotic systems (such asOROS® (Alza Corporation, Mountain View, Calif. USA)), or a combinationthereof to provide the desired release profile in varying proportions.

In some embodiments of any of the aspects, a composition or combinationdescribed herein is administered as a monotherapy, e.g., anothertreatment for the cancer is not administered to the subject.

In some embodiments of any of the aspects, the methods described hereincan further comprise administering a second agent and/or treatment tothe subject, e.g. as part of a combinatorial therapy. Non-limitingexamples of a second agent and/or treatment can include radiationtherapy, surgery, gemcitabine, cisplastin, paclitaxel, carboplatin,bortezomib, AMG479, vorinostat, ntuximab, temozolomide, rapamycin,ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN®cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegaI1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); lapatinib (Tykerb®); inhibitors of PKC-alpha, Raf,H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cellproliferation and pharmaceutically acceptable salts, acids orderivatives of any of the above.

In addition, the methods of treatment can further include the use ofradiation or radiation therapy. Further, the methods of treatment canfurther include the use of surgical treatments.

In some embodiments of any of the aspects, the method of treatmentdescribed herein further comprises administering a BET inhibitor. Insome embodiments of any of the aspects, described herein is thecombination of a) an exosome, inhibitor, and/or agonist and b) a BETinhibitor, for use in treating cancer. Bromodomain and Extra-Terminaldomain (BET) family proteins are epigenetic regulatory factors relatedto the expression of multiple oncogenes. BET inhibitors are a class ofdrugs that reversibly bind the bromodomains of BET proteins and preventprotein-protein interaction between BET proteins and acetylated histonesand transcription factors to control DNA transcription. Examples of BETinhibitors include but are not limited to: ABBV-075, ABBV-744,BAY1238097, BI 894999, BMS-986158, CPI-203, CPI-0610, CPI-1205, FT-1101,GS-5829, GSK-046, GSK-726, GSK525762, I-BET762, GSK525762A, GSK2820151,I-BET151, INCB054329, OTX015/MK-8628, PLX51107, RO6870810, TEN-010,ZEN003694, CPI-0610, JQ1, RVX-208, MS417, SJ432, AZD5153, INCB054329,RVX000222, ARV825, BIC1, ZEN-3694, BET-IN-6, BET-BAY 002 (S enantiomer),BET-BAY 002, BET bromodomain inhibitor 1, BET-IN-1, BET-IN-2, BETbromodomain inhibitor, ODM-207, BETd-246, BETd-260, I-BET151dihydrochloride, NVS-BET-1, I-BET567, Molibresib besylate, I-BET282,I-BET282E, Molibresib, HDAC/BET-IN-1, I-BET762 carboxylic acid, GSK040,GSK1324726A, OXFBD04, (S)-GNE-987, Amredobresib, INCB054329 Racemate,INCB-057643, CF53, Desmethyl-QCA276, (+)-JQ1 PA, Bromodomaininhibitor-8, NE02734, CD235, (Rac)-BAY1238097, GSK778, PFI-1, (S)-JQ-35,GSK852, CPI-203, Trotabresib, HJB97, Y06036, PNZ5, Y06137,(+)-JQ-1-aldehyde, BRD4 D1-IN-1,BRD4 D1-IN-2, GS-626510, (+)-JQ-1, BY27,BMS-986158, AZD5153 6-Hydroxy-2-naphthoic acid, GSK097, BI-9564, MS645,CD161, ZEN-3862, MS417, (R)-BAY1238097, PLX51107, GSK620, LT052,NHWD-870, SNIPER(BRD)-1, ARV-771, ZEN-3411, ZEN-3219, RVX-297, MS402,and GSK973.

In some embodiments of any of the aspects, the method of treatmentdescribed herein further comprises administering a PROTAC degrader. Insome embodiments of any of the aspects, described herein is thecombination of a) an exosome, inhibitor, and/or agonist and b) a PROTACdegrader, for use in treating cancer. Proteolysis Targeting Chimeric(PROTAC) technology is an endogenous protein degradation tool thatutilizes ubiquitin and targets tumor proteins by using theubiquitin-proteosome system (UPS) to ubiquitinate and degrade tumorproteins, achieving an effect on tumor growth. Through the use of aheterobifunctional small molecule consisting of two ligands joined by alinker, one ligand recruits and binds to the tumor protein of interest(POI) while the other recruits and binds and E3 ubiquitin ligase. Thisresults in the tumor POI's subsequent degradation through the UPS andthe PROTAC is recycled to target another tumor POI. Examples of PROTACdegraders include but are not limited to: ARV-110, ARV-471, dBET1, DT-6,CP-10, C3, C5, Compounds 6A-D, SD-36, BETd-260, PROTAC7, CP5V, ARD-61,ARD-266, Compound I-6, Compound 3, SIAIS178, UNC6852, A1874, Compounds4, β-NF-ATRA, β-NF-JQ1, KT-474, NX-2127, DT2216, METAP2, AC682, ARV-766,CC-94676, FHD-609, KT-413, KT-333, NX-5948, CFT8634, CFT8919, CG001419,CC-220, CC-92480, CC-90009, CC-99282, CFT7455, DKY709, VZ185, MZ1,ARV-825, dBET6, dBET1, ARV-771, AU-15330, PROTAC CBP/P300 Degrader-1,dBET23, LC-2, ACBI1, Gefitinib-based PROTAC 3, BSJ-03-123, SD-36,MD-224, THAL-SNS-032, BETd-260, BRD4 degrader AT1, dTRIM24, BSJ-4-116,dBET57, ARCC-4, MT-802, UNC6852, SJF620, ZXH-3-26, A1874, PROTAC FAKdegrader 1, CP-10, BSJ-03-204, XZ739, PROTAC Mcl1 degrader-1, GNE-987,MS4078, ARD-266, PROTAC SGK3 degrader-1, dCBP-1, PROTAC RIPK degrader-2,FKBP12 PROTAC RC32, dFKBP-1, BI-3663, PROTAC FLT-3 degrader 1, PROTACBcl2 degrader-1, PROTAC CDK9 Degrader-1, PROTAC B-Raf degrader 1, FKBP12PROTAC dTAG-13, TL12-186, PROTAC Sirt2 Degrader-1, BSJ-04-132, PROTACRIPK degrader-6, PROTAC BRD9 Degrader-1, JH-XI-10-02, ERD-308,Homo-PROTAC pVHL30 degrader 1, PROTAC CDK2/9 Degrader-1, SIAIS178,XY028-140, MZP-55, PROTAC ERα Degrader-2, GMB-475, BETd-246, MZP-54,PROTAC IRAK4 degrader-1, PROTAC CRBN Degrader-1, PROTAC FKBP Degrader-3,KB02-JQ1, MS4077, PROTAC AR Degrader-4 TFA, PROTAC MDM2 Degrader-3,PROTAC ERα Degrader-1, PROTAC BET Degrader-1, Homo-PROTAC cereblondegrader 1, SJF620 hydrochloride, PROTAC BET Degrader-10, PROTAC EEDdegrader-2, JB170, CP5V, KB02-SLF, XY028-133, PROTAC BET degrader-2,PROTAC RAR Degrader-1, QCA570, PROTAC BET degrader-3, PROTAC EEDdegrader-1, AT6, MS432, PROTAC BRD4 Degrader-5, MD-222, PROTAC BRD4Degrader-8, TD-165, PROTAC MDM2 Degrader-1, ARD-2585, PROTAC KRAS G12Cdegrader-1, PROTAC ERRα Degrader-3, SIM1, PROTAC BRD4 Degrader-9, PROTACPD-1/PD-L1 degrader-1, AGB1, PROTAC-O4I2, PROTAC IDO1 Degrader-1, PROTACER Degrader-4, ARD-2128, PROTAC AR Degrader-4, FKBP12 PROTAC dTAG-7,PROTAC PARP1 degrader, ZXH-4-130 TFA, PZ703b, PROTAC BRD4 Degrader-2,PROTAC BRD4 Degrader-1, PROTAC ERRα Degrader-2, MS67, PROTAC CRABP-IIDegrader-2, PROTAC BRD4 Degrader-7, PROTAC BRD2/BRD4 degrader-1, PROTACBcl-xL degrader-3, PROTAC BRD4 Degrader-11, XY-06-007, SJFδ, PROTAC BRD4Degrader-12, dTAGV-1 TFA, SHP2-D26, MS4322, TL13-12, PROTAC MDM2Degrader-4, PROTAC Bcl-xL degrader-2, PROTAC ER Degrader-3, PROTACCRABP-II Degrader-3, GSK215, PROTAC BRD9 Degrader-4, PROTAC BRD4Degrader-14, PROTAC BRD4 Degrader-10, OARV-771, PROTAC CRABP-IIDegrader-1, PROTAC ERRα Degrader-1, PROTAC Bcl-xL degrader-1,(S)-GNE-987, CRBN-6-5-5-VHL, CCT367766, PROTAC MDM2 Degrader-2,CFT-2718, TD-428, PROTAC BRD4 Degrader-3, Thalidomide-NH-CBP/p300 ligand2, dMCL1-2, SJFα, MS170, PROTAC BRD4 Degrader-13, PROTAC ER Degrader-10,TL13-112, PROTAC IRAK4 degrader-3, INY-03-041, PROTAC ER Degrader-2,DP-C-4, RIP2 Kinase Inhibitor 4, SJ10542, MS98, PhosTAC7, PROTAC BRD9Degrader-2, PROTAC IRAK4 degrader-5, MS33, PROTAC CDK9 degrader-2, MS21,Dovitinib-RIBOTAC, SHP2 protein degrader-1, ZXH-4-130, PROTAC IRAK3degrade-1, MS83, PROTAC IRAK4 degrader-4, Folate-MS432, PROTAC BRD4Degrader-15, PROTAC BRD9 Degrader-3, PROTAC IRAK4 degrader-6, PROTACCDK9 degrader-4, Pomalidomide-C5-Dovitinib, ARD-61, ZXH-4-137, CMP98,di-Ellipticine-RIBOTAC, CC-885-CH2-PEG1-NH-CH3, SJ995973, HDAC6degrader-1.

In certain embodiments, an effective dose of a composition orcombination as described herein can be administered to a patient once.In certain embodiments, an effective dose of a composition orcombination as described herein can be administered to a patientrepeatedly. For systemic administration, subjects can be administered atherapeutic amount of a composition or combination as described herein,such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50mg/kg, or more.

In some embodiments, after an initial treatment regimen, the treatmentscan be administered on a less frequent basis. For example, aftertreatment biweekly for three months, treatment can be repeated once permonth, for six months or a year or longer. Treatment according to themethods described herein can reduce levels of a marker or symptom of acondition, e.g. tumor size or growth, or EMT rate or prevelance by atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80% or atleast 90% or more.

The dosage of a composition as described herein can be determined by aphysician and adjusted, as necessary, to suit observed effects of thetreatment. With respect to duration and frequency of treatment, it istypical for skilled clinicians to monitor subjects in order to determinewhen the treatment is providing therapeutic benefit, and to determinewhether to increase or decrease dosage, increase or decreaseadministration frequency, discontinue treatment, resume treatment, ormake other alterations to the treatment regimen. The dosing schedule canvary from once a week to daily depending on a number of clinicalfactors, such as the subject's sensitivity to the active ingredient(s).The desired dose or amount of activation can be administered at one timeor divided into subdoses, e.g., 2-4 subdoses and administered over aperiod of time, e.g., at appropriate intervals through the day or otherappropriate schedule. In some embodiments, administration can bechronic, e.g., one or more doses and/or treatments daily over a periodof weeks or months. Examples of dosing and/or treatment schedules areadministration daily, twice daily, three times daily or four or moretimes daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month,2 months, 3 months, 4 months, 5 months, or 6 months, or more. Acomposition or combination as described herein can be administered overa period of time, such as over a 5 minute, 10 minute, 15 minute, 20minute, or 25 minute period.

The dosage ranges for the administration of a composition or combinationas described herein, according to the methods described herein dependupon, for example, the form of the composition or combination asdescribed herein, its potency, and the extent to which symptoms,markers, or indicators of a condition described herein are desired to bereduced, for example the percentage reduction desired for tumor size,tumor growth, or EMT rate or prevelance. The dosage should not be solarge as to cause adverse side effects. Generally, the dosage will varywith the age, condition, and sex of the patient and can be determined byone of skill in the art. The dosage can also be adjusted by theindividual physician in the event of any complication.

The efficacy of composition or combination as described herein in, e.g.the treatment of a condition described herein, or to induce a responseas described herein can be determined by the skilled clinician. However,a treatment is considered “effective treatment,” as the term is usedherein, if one or more of the signs or symptoms of a condition describedherein are altered in a beneficial manner, other clinically acceptedsymptoms are improved, or even ameliorated, or a desired response isinduced e.g., by at least 10% following treatment according to themethods described herein. Efficacy can be assessed, for example, bymeasuring a marker, indicator, symptom, and/or the incidence of acondition treated according to the methods described herein or any othermeasurable parameter appropriate, e.g. EMT marker genes. Efficacy canalso be measured by a failure of an individual to worsen as assessed byhospitalization, or need for medical interventions (i.e., progression ofthe disease is halted). Methods of measuring these indicators are knownto those of skill in the art and/or are described herein. Treatmentincludes any treatment of a disease in an individual or an animal (somenon-limiting examples include a human or an animal) and includes: (1)inhibiting the disease, e.g., preventing a worsening of symptoms (e.g.pain or inflammation); or (2) relieving the severity of the disease,e.g., causing regression of symptoms. An effective amount for thetreatment of a disease means that amount which, when administered to asubject in need thereof, is sufficient to result in effective treatmentas that term is defined herein, for that disease. Efficacy of an agentcan be determined by assessing physical indicators of a condition ordesired response, e.g., tumor size, tumor growth, or EMT rate orprevelance. It is well within the ability of one skilled in the art tomonitor efficacy of administration and/or treatment by measuring any oneof such parameters, or any combination of parameters. Efficacy can beassessed in animal models of a condition described herein, for exampletreatment of cancer. When using an experimental animal model, efficacyof treatment is evidenced when a statistically significant change in amarker is observed, e.g. EMT marker genes described elsewhere herein.

In one respect, the present invention relates to the herein describedcompositions, methods, and respective component(s) thereof, as essentialto the technology, yet open to the inclusion of unspecified elements,essential or not (“comprising). In some embodiments of any of theaspects, other elements to be included in the description of thecomposition, method or respective component thereof are limited to thosethat do not materially affect the basic and novel characteristic(s) ofthe technology (e.g., the composition, method, or respective componentthereof “consists essentially of” the elements described herein). Thisapplies equally to steps within a described method as well ascompositions and components therein. In other embodiments of any of theaspects, the compositions, methods, and respective components thereof,described herein are intended to be exclusive of any element not deemedan essential element to the component, composition or method (e.g., thecomposition, method, or respective component thereof “consists of” theelements described herein). This applies equally to steps within adescribed method as well as compositions and components therein.

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed invention, because the scope of theinvention is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. If there is an apparent discrepancy between the usageof a term in the art and its definition provided herein, the definitionprovided within the specification shall prevail.

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here.

The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all usedherein to mean a decrease by a statistically significant amount. In someembodiments, “reduce,” “reduction” or “decrease” or “inhibit” typicallymeans a decrease by at least 10% as compared to a reference level (e.g.the absence of a given treatment or agent) and can include, for example,a decrease by at least about 10%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, at least about 99% , or more. As used herein,“reduction” or “inhibition” does not encompass a complete inhibition orreduction as compared to a reference level. “Complete inhibition” is a100% inhibition as compared to a reference level. A decrease can bepreferably down to a level accepted as within the range of normal for anindividual without a given disorder.

The terms “increased”, “increase”, “enhance”, or “activate” are all usedherein to mean an increase by a statically significant amount. In someembodiments, the terms “increased”, “increase”, “enhance”, or “activate”can mean an increase of at least 10% as compared to a reference level,for example an increase of at least about 20%, or at least about 30%, orat least about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or up toand including a 100% increase or any increase between 10-100% ascompared to a reference level, or at least about a 2-fold, or at leastabout a 3-fold, or at least about a 4-fold, or at least about a 5-foldor at least about a 10-fold increase, or any increase between 2-fold and10-fold or greater as compared to a reference level. In the context of amarker or symptom, a “increase” is a statistically significant increasein such level.

In some embodiments of any of the aspects, an increased level ofexpression, e.g., one which indicates a patient is high or increasedrisk or is in need of a treatment as described herein is 1.25 fold orgreater change relative to a reference. In some embodiments of any ofthe aspects, an increased level of expression, e.g., one which indicatesa patient is high or increased risk or is in need of a treatment asdescribed herein is 2.5 fold or greater change relative to a reference.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologus monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Insome embodiments, the subject is a mammal, e.g., a primate, e.g., ahuman. The terms, “individual,” “patient” and “subject” are usedinterchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of cancer.A subject can be male or female.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a condition in need of treatment(e.g. cancer) or one or more complications related to such a condition,and optionally, have already undergone treatment for the condition orthe one or more complications related to the condition. Alternatively, asubject can also be one who has not been previously diagnosed as havingthe condition or one or more complications related to the condition. Forexample, a subject can be one who exhibits one or more risk factors forthe condition or one or more complications related to the condition or asubject who does not exhibit risk factors.

A “subject in need” of treatment for a particular condition can be asubject having that condition, diagnosed as having that condition, or atrisk of developing that condition.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably herein to designate a series of amino acid residues,connected to each other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and“polypeptide” refer to a polymer of amino acids, including modifiedamino acids (e.g., phosphorylated, glycated, glycosylated, etc.) andamino acid analogs, regardless of its size or function. “Protein” and“polypeptide” are often used in reference to relatively largepolypeptides, whereas the term “peptide” is often used in reference tosmall polypeptides, but usage of these terms in the art overlaps. Theterms “protein” and “polypeptide” are used interchangeably herein whenreferring to a gene product and fragments thereof Thus, exemplarypolypeptides or proteins include gene products, naturally occurringproteins, homologs, orthologs, paralogs, fragments and otherequivalents, variants, fragments, and analogs of the foregoing. Theterms also refer to fragments or variants of the polypeptide thatmaintain at least 50% of the activity or effect of the full lengthreference polypeptide. Conservative substitution variants that maintainthe activity of a wildtype protein will include a conservativesubstitution as defined herein. The identification of amino acids mostlikely to be tolerant of conservative substitution while maintaining atleast 50% of the activity of the wildtype is guided by, for example,sequence alignment with homologs or paralogs from other species. Aminoacids that are identical between homologs are less likely to toleratechange, while those showing conservative differences are obviously muchmore likely to tolerate conservative change in the context of anartificial variant. Similarly, positions with non-conservativedifferences are less likely to be critical to function and more likelyto tolerate conservative substitution in an artificial variant.Variants, fragments, and/or fusion proteins can be tested for activity,for example, by administering the variant to an appropriate animal modelof cancer as described herein.

In some embodiments, a polypeptide can be a variant of a sequencedescribed herein. In some embodiments, the variant is a conservativesubstitution variant. Variants can be obtained by mutations of nativenucleotide sequences, for example. A “variant,” as referred to herein,is a polypeptide substantially homologous to a native or referencepolypeptide, but which has an amino acid sequence different from that ofthe native or reference polypeptide because of one or a plurality ofdeletions, insertions or substitutions. Polypeptide-encoding DNAsequences encompass sequences that comprise one or more additions,deletions, or substitutions of nucleotides when compared to a native orreference DNA sequence, but that encode a variant protein or fragmentthereof that retains the relevant biological activity relative to thereference protein, e.g., at least 50% of that displayed by the wildtype.As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters a single amino acid or asmall percentage, (i.e. 5% or fewer, e.g. 4% or fewer, or 3% or fewer,or 1% or fewer) of amino acids in the encoded sequence is a“conservatively modified variant” where the alteration results in thesubstitution of an amino acid with a chemically similar amino acid. Itis contemplated that some changes can potentially improve the relevantactivity, such that a variant, whether conservative or not, has morethan 100% of the activity of a wildtype protein, e.g. 110%, 125%, 150%,175%, 200%, 500%, 1000% or more.

One method of identifying amino acid residues which can be substitutedis to align, for example, a human protein to a homolog from one or morenon-human species. Alignment can provide guidance regarding not onlyresidues likely to be necessary for function but also, conversely, thoseresidues likely to tolerate change. Where, for example, an alignmentshows two identical or similar amino acids at corresponding positions,it is more likely that that site is important functionally. Where,conversely, alignment shows residues in corresponding positions todiffer significantly in size, charge, hydrophobicity, etc., it is morelikely that that site can tolerate variation in a functionalpolypeptide. The variant amino acid or DNA sequence can be at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, ormore, identical to a native or reference sequence, or a nucleic acidencoding one of those amino acid sequences. The degree of homology(percent identity) between a native and a mutant sequence can bedetermined, for example, by comparing the two sequences using freelyavailable computer programs commonly employed for this purpose on theworld wide web. The variant amino acid or DNA sequence can be at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or more,similar to the sequence from which it is derived (referred to herein asan “original” sequence). The degree of similarity (percent similarity)between an original and a mutant sequence can be determined, forexample, by using a similarity matrix. Similarity matrices are wellknown in the art and a number of tools for comparing two sequences usingsimilarity matrices are freely available online, e.g. BLASTp or BLASTn(available on the world wide web at blast.ncbi.nlm.nih.gov), withdefault parameters set.

In the various embodiments described herein, it is further contemplatedthat variants (naturally occurring or otherwise), alleles, homologs,conservatively modified variants, and/or conservative substitutionvariants of any of the particular polypeptides described areencompassed. As to amino acid sequences, one of skill will recognizethat individual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters a single aminoacid or a small percentage of amino acids in the encoded sequence is a“conservatively modified variant” where the alteration results in thesubstitution of an amino acid with a chemically similar amino acid andretains the desired activity of the polypeptide. Such conservativelymodified variants are in addition to and do not exclude polymorphicvariants, interspecies homologs, and alleles consistent with thedisclosure.

A given amino acid can be replaced by a residue having similarphysiochemical characteristics, e.g., substituting one aliphatic residuefor another (such as Ile, Val, Leu, or Ala for one another), orsubstitution of one polar residue for another (such as between Lys andArg; Glu and Asp; or Gln and Asn). Other such conservativesubstitutions, e.g., substitutions of entire regions having similarhydrophobicity characteristics, are well known. Polypeptides comprisingconservative amino acid substitutions can be tested in any one of theassays described herein to confirm that a desired activity andspecificity of a native or reference polypeptide is retained.

A given amino acid can be replaced by a residue having similarphysiochemical characteristics, e.g., substituting one aliphatic residuefor another (such as Ile, Val, Leu, or Ala for one another), orsubstitution of one polar residue for another (such as between Lys andArg; Glu and Asp; or Gln and Asn). Other such conservativesubstitutions, e.g., substitutions of entire regions having similarhydrophobicity characteristics, are well known. Polypeptides comprisingconservative amino acid substitutions can be tested in any one of theassays described herein to confirm that a desired activity of a nativeor reference polypeptide is retained. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and allelesconsistent with the disclosure.

Amino acids can be grouped according to similarities in the propertiesof their side chains (in A. L. Lehninger, in Biochemistry, second ed.,pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A),Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2)uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N),Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His(H). Alternatively, naturally occurring residues can be divided intogroups based on common side-chain properties: (1) hydrophobic:Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser,Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5)residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp,Tyr, Phe. Non-conservative substitutions will entail exchanging a memberof one of these classes for another class. Particular conservativesubstitutions include, for example; Ala into Gly or into Ser; Arg intoLys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln into Asn;Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ileinto Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Glnor into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leuor into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp;and/or Phe into Val, into Ile or into Leu. Typically conservativesubstitutions for one another also include: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

In some embodiments, the polypeptide described herein (or a nucleic acidencoding such a polypeptide) can be a functional fragment of one of theamino acid sequences described herein. As used herein, a “functionalfragment” is a fragment or segment of a peptide which retains at least50% of the wildtype reference polypeptide's activity according to theassays described below herein. A functional fragment can compriseconservative substitutions of the sequences disclosed herein.

In some embodiments, the polypeptide described herein can be a variantof a sequence described herein. In some embodiments, the variant is aconservatively modified variant. Conservative substitution variants canbe obtained by mutations of native nucleotide sequences, for example. A“variant,” as referred to herein, is a polypeptide substantiallyhomologous to a native or reference polypeptide, but which has an aminoacid sequence different from that of the native or reference polypeptidebecause of one or a plurality of deletions, insertions or substitutions.Variant polypeptide-encoding DNA sequences encompass sequences thatcomprise one or more additions, deletions, or substitutions ofnucleotides when compared to a native or reference DNA sequence, butthat encode a variant protein or fragment thereof that retains activity.A wide variety of PCR-based site-specific mutagenesis approaches areknown in the art and can be applied by the ordinarily skilled artisan.

In some embodiments, a polypeptide, e can comprise one or more aminoacid substitutions or modifications. In some embodiments, thesubstitutions and/or modifications can prevent or reduce proteolyticdegradation and/or prolong half-life of the polypeptide in a subject. Insome embodiments, a polypeptide can be modified by conjugating or fusingit to other polypeptide or polypeptide domains such as, by way ofnon-limiting example, transferrin (W006096515A2), albumin (Yeh et al.,1992), growth hormone (US2003104578AA); cellulose (Levy and Shoseyov,2002); and/or Fc fragments (Ashkenazi and Chamow, 1997). The referencesin the foregoing paragraph are incorporated by reference herein in theirentireties.

In some embodiments, a polypeptide as described herein can comprise atleast one peptide bond replacement. A polypeptide as described hereincan comprise one type of peptide bond replacement or multiple types ofpeptide bond replacements, e.g. 2 types, 3 types, 4 types, 5 types, ormore types of peptide bond replacements. Non-limiting examples ofpeptide bond replacements include urea, thiourea, carbamate, sulfonylurea, trifluoroethylamine, ortho-(aminoalkyl)-phenylacetic acid,para-(aminoalkyl)-phenylacetic acid, meta-(aminoalkyl)-phenylaceticacid, thioamide, tetrazole, boronic ester, olefinic group, andderivatives thereof.

In some embodiments, a polypeptide as described herein can comprisenaturally occurring amino acids commonly found in polypeptides and/orproteins produced by living organisms, e.g. Ala (A), Val (V), Leu (L),Ile (I), Pro (P), Phe (F), Trp (W), Met (M), Gly (G), Ser (S), Thr (T),Cys (C), Tyr (Y), Asn (N), Gln (Q), Asp (D), Glu (E), Lys (K), Arg (R),and His (H). In some embodiments, a polypeptide as described herein cancomprise alternative amino acids. Non-limiting examples of alternativeamino acids include, D-amino acids; beta-amino acids; homocysteine,phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline,gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylicacid, statine, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid,penicillamine (3-mercapto-D-valine), ornithine, citruline,alpha-methyl-alanine, para-benzoylphenylalanine, para-aminophenylalanine, p-fluorophenylalanine, phenylglycine, propargylglycine,sarcosine, and tert-butylglycine), diaminobutyric acid,7-hydroxy-tetrahydroisoquinoline carboxylic acid, naphthylalanine,biphenylalanine, cyclohexylalanine, amino-isobutyric acid, norvaline,norleucine, tert-leucine, tetrahydroisoquinoline carboxylic acid,pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine,dehydroleucine, 2,2-diethylglycine, 1-amino-l-cyclopentanecarboxylicacid, 1-amino-l-cyclohexanecarboxylic acid, amino-benzoic acid,amino-naphthoic acid, gamma-aminobutyric acid, difluorophenylalanine,nipecotic acid, alpha-amino butyric acid, thienyl-alanine,t-butylglycine, trifluorovaline; hexafluoroleucine; fluorinated analogs;azide-modified amino acids; alkyne-modified amino acids; cyano-modifiedamino acids; and derivatives thereof.

In some embodiments, a polypeptide can be modified, e.g. by addition ofa moiety to one or more of the amino acids that together comprise thepeptide. In some embodiments, a polypeptide as described herein cancomprise one or more moiety molecules, e.g. 1 or more moiety moleculesper polypeptide, 2 or more moiety molecules per polypeptide, 5 or moremoiety molecules per polypeptide, 10 or more moiety molecules perpolypeptide or more moiety molecules per polypeptide. In someembodiments, a polypeptide as described herein can comprise one moretypes of modifications and/or moieties, e.g. 1 type of modification, 2types of modifications, 3 types of modifications or more types ofmodifications. Non-limiting examples of modifications and/or moietiesinclude PEGylation; glycosylation; HESylation; ELPylation; lipidation;acetylation; amidation; end-capping modifications; cyano groups;phosphorylation; albumin, and cyclization. In some embodiments, anend-capping modification can comprise acetylation at the N-terminus,N-terminal acylation, and N-terminal formylation. In some embodiments,an end-capping modification can comprise amidation at the C-terminus,introduction of C-terminal alcohol, aldehyde, ester, and thioestermoieties. The half-life of a polypeptide can be increased by theaddition of moieties, e.g. PEG, albumin, or other fusion partners (e.g.Fc fragment of an immunoglobin).

Any cysteine residue not involved in maintaining the proper conformationof the polypeptide also can be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) can be added to thepolypeptide to improve its stability or facilitate oligomerization.

Alterations of the native amino acid sequence can be accomplished by anyof a number of techniques known to one of skill in the art. Mutationscan be introduced, for example, at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered nucleotide sequencehaving particular codons altered according to the substitution,deletion, or insertion required. Techniques for making such alterationsare very well established. Alterations of the original amino acidsequence can be accomplished by any of a number of techniques known toone of skill in the art. Mutations can be introduced, for example, atparticular loci by synthesizing oligonucleotides containing a mutantsequence, flanked by restriction sites permitting ligation to fragmentsof the native sequence. Following ligation, the resulting reconstructedsequence encodes an analog having the desired amino acid insertion,substitution, or deletion. Alternatively, oligonucleotide-directedsite-specific mutagenesis procedures can be employed to provide analtered nucleotide sequence having particular codons altered accordingto the substitution, deletion, or insertion required. Techniques formaking such alterations include those disclosed by Khudyakov et al.“Artificial DNA: Methods and Applications” CRC Press, 2002; Braman “InVitro Mutagenesis Protocols” Springer, 2004; and Rapley “The NucleicAcid Protocols Handbook” Springer 2000; which are herein incorporated byreference in their entireties. In some embodiments, a polypeptide asdescribed herein can be chemically synthesized and mutations can beincorporated as part of the chemical synthesis process.

As used herein, the term “nucleic acid” or “nucleic acid sequence”refers to any molecule, preferably a polymeric molecule, incorporatingunits of ribonucleic acid, deoxyribonucleic acid or an analog thereofThe nucleic acid can be either single-stranded or double-stranded. Asingle-stranded nucleic acid can be one nucleic acid strand of adenatured double- stranded DNA. Alternatively, it can be asingle-stranded nucleic acid not derived from any double-stranded DNA.In one aspect, the nucleic acid can be DNA. In another aspect, thenucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA orcDNA. Suitable RNA can include, e.g., mRNA.

The term “expression” refers to the cellular processes involved inproducing RNA and proteins and as appropriate, secreting proteins,including where applicable, but not limited to, for example,transcription, transcript processing, translation and protein folding,modification and processing. Expression can refer to the transcriptionand stable accumulation of sense (mRNA) or antisense RNA derived from anucleic acid fragment or fragments of the invention and/or to thetranslation of mRNA into a polypeptide.

In some embodiments, the expression of a biomarker(s), target(s), orgene/polypeptide described herein is/are tissue-specific. In someembodiments, the expression of a biomarker(s), target(s), orgene/polypeptide described herein is/are global. In some embodiments,the expression of a biomarker(s), target(s), or gene/polypeptidedescribed herein is systemic.

“Expression products” include RNA transcribed from a gene, andpolypeptides obtained by translation of mRNA transcribed from a gene.The term “gene” means the nucleic acid sequence which is transcribed(DNA) to RNA in vitro or in vivo when operably linked to appropriateregulatory sequences. The gene may or may not include regions precedingand following the coding region, e.g. 5′ untranslated (5′UTR) or“leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons).

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, control elements operably linked to a coding sequenceare capable of effecting the expression of the coding sequence. Thecontrol elements need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter sequence and the coding sequence and thepromoter sequence can still be considered “operably linked” to thecoding sequence.

“Marker” in the context of the present invention refers to an expressionproduct, e.g., nucleic acid or polypeptide which is differentiallypresent in a sample taken from subjects having having diabetes orcancer, as compared to a comparable sample taken from control subjects(e.g., a healthy subject). The term “biomarker” is used interchangeablywith the term “marker.”

In some embodiments, the methods described herein relate to measuring,detecting, or determining the level of at least one marker. As usedherein, the term “detecting” or “measuring” refers to observing a signalfrom, e.g. a probe, label, or target molecule to indicate the presenceof an analyte in a sample. Any method known in the art for detecting aparticular label moiety can be used for detection. Exemplary detectionmethods include, but are not limited to, spectroscopic, fluorescent,photochemical, biochemical, immunochemical, electrical, optical orchemical methods. In some embodiments of any of the aspects, measuringcan be a quantitative observation.

In some embodiments of any of the aspects, a polypeptide, nucleic acid,or cell as described herein can be engineered. As used herein,“engineered” refers to the aspect of having been manipulated by the handof man. For example, a polypeptide is considered to be “engineered” whenat least one aspect of the polypeptide, e.g., its sequence, has beenmanipulated by the hand of man to differ from the aspect as it exists innature. As is common practice and is understood by those in the art,progeny of an engineered cell are typically still referred to as“engineered” even though the actual manipulation was performed on aprior entity.

In some embodiments of any of the aspects, the inhibitor, agonist, orexosome administered to a subject as described herein is exogenous. Insome embodiments of any of the aspects, the inhibitor, agonist, orexosome administered to a subject as described herein is ectopic. Insome embodiments of any of the aspects, the inhibitor, agonist, orexosome administered to a subject as described herein is not endogenous.

The term “exogenous” refers to a substance present in a cell other thanits native source. The term “exogenous” when used herein can refer to anucleic acid (e.g. a nucleic acid encoding a polypeptide) or apolypeptide that has been introduced by a process involving the hand ofman into a biological system such as a cell or organism in which it isnot normally found and one wishes to introduce the nucleic acid orpolypeptide into such a cell or organism. Alternatively, “exogenous” canrefer to a nucleic acid or a polypeptide that has been introduced by aprocess involving the hand of man into a biological system such as acell or organism in which it is found in relatively low amounts and onewishes to increase the amount of the nucleic acid or polypeptide in thecell or organism, e.g., to create ectopic expression or levels. Incontrast, the term “endogenous” refers to a substance that is native tothe biological system or cell. As used herein, “ectopic” refers to asubstance that is found in an unusual location and/or amount. An ectopicsubstance can be one that is normally found in a given cell, but at amuch lower amount and/or at a different time. Ectopic also includessubstance, such as a polypeptide or nucleic acid that is not naturallyfound or expressed in a given cell in its natural environment.

In some embodiments, a nucleic acid encoding a polypeptide as describedherein (e.g. an agonist polypeptide) is comprised by a vector. In someof the aspects described herein, a nucleic acid sequence encoding agiven polypeptide as described herein, or any module thereof, isoperably linked to a vector. The term “vector”, as used herein, refersto a nucleic acid construct designed for delivery to a host cell or fortransfer between different host cells. As used herein, a vector can beviral or non-viral. The term “vector” encompasses any genetic elementthat is capable of replication when associated with the proper controlelements and that can transfer gene sequences to cells. A vector caninclude, but is not limited to, a cloning vector, an expression vector,a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.

In some embodiments of any of the aspects, the vector is recombinant,e.g., it comprises sequences originating from at least two differentsources. In some embodiments of any of the aspects, the vector comprisessequences originating from at least two different species. In someembodiments of any of the aspects, the vector comprises sequencesoriginating from at least two different genes, e.g., it comprises afusion protein or a nucleic acid encoding an expression product which isoperably linked to at least one non-native (e.g., heterologous) geneticcontrol element (e.g., a promoter, suppressor, activator, enhancer,response element, or the like).

In some embodiments of any of the aspects, the vector or nucleic aciddescribed herein is codon-optomized, e.g., the native or wild-typesequence of the nucleic acid sequence has been altered or engineered toinclude alternative codons such that altered or engineered nucleic acidencodes the same polypeptide expression product as the native/wild-typesequence, but will be transcribed and/or translated at an improvedefficiency in a desired expression system. In some embodiments of any ofthe aspects, the expression system is an organism other than the sourceof the native/wild-type sequence (or a cell obtained from suchorganism). In some embodiments of any of the aspects, the vector and/ornucleic acid sequence described herein is codon-optimized for expressionin a mammal or mammalian cell, e.g., a mouse, a murine cell, or a humancell. In some embodiments of any of the aspects, the vector and/ornucleic acid sequence described herein is codon-optimized for expressionin a human cell. In some embodiments of any of the aspects, the vectorand/or nucleic acid sequence described herein is codon-optimized forexpression in a yeast or yeast cell. In some embodiments of any of theaspects, the vector and/or nucleic acid sequence described herein iscodon-optimized for expression in a bacterial cell. In some embodimentsof any of the aspects, the vector and/or nucleic acid sequence describedherein is codon-optimized for expression in an E. coli cell.

As used herein, the term “expression vector” refers to a vector thatdirects expression of an RNA or polypeptide from sequences linked totranscriptional regulatory sequences on the vector. The sequencesexpressed will often, but not necessarily, be heterologous to the cell.An expression vector may comprise additional elements, for example, theexpression vector may have two replication systems, thus allowing it tobe maintained in two organisms, for example in human cells forexpression and in a prokaryotic host for cloning and amplification.

As used herein, the term “viral vector” refers to a nucleic acid vectorconstruct that includes at least one element of viral origin and has thecapacity to be packaged into a viral vector particle. The viral vectorcan contain the nucleic acid encoding a polypeptide as described hereinin place of non-essential viral genes. The vector and/or particle may beutilized for the purpose of transferring any nucleic acids into cellseither in vitro or in vivo. Numerous forms of viral vectors are known inthe art.

It should be understood that the vectors described herein can, in someembodiments, be combined with other suitable compositions and therapies.In some embodiments, the vector is episomal. The use of a suitableepisomal vector provides a means of maintaining the nucleotide ofinterest in the subject in high copy number extra chromosomal DNAthereby eliminating potential effects of chromosomal integration.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with a disease ordisorder, e.g. cancer. The term “treating” includes reducing oralleviating at least one adverse effect or symptom of a condition,disease or disorder associated with a cancer. Treatment is generally“effective” if one or more symptoms or clinical markers are reduced.Alternatively, treatment is “effective” if the progression of a diseaseis reduced or halted. That is, “treatment” includes not just theimprovement of symptoms or markers, but also a cessation of, or at leastslowing of, progress or worsening of symptoms compared to what would beexpected in the absence of treatment. Beneficial or desired clinicalresults include, but are not limited to, alleviation of one or moresymptom(s), diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, remission (whetherpartial or total), and/or decreased mortality, whether detectable orundetectable. The term “treatment” of a disease also includes providingrelief from the symptoms or side-effects of the disease (includingpalliative treatment).

In some embodiments of any of the aspects, described herein is aprophylactic method of treatment. As used herein “prophylactic” refersto the timing and intent of a treatment relative to a disease orsymptom, that is, the treatment is administered prior to clinicaldetection or diagnosis of that particular disease or symptom in order toprotect the patient from the disease or symptom. Prophylactic treatmentcan encompass a reduction in the severity or speed of onset of thedisease or symptom, or contribute to faster recovery from the disease orsymptom. Accordingly, the methods described herein can be prophylacticrelative to metastasis or tumor formation. In some embodiments of any ofthe aspects, prophylactic treatment is not prevention of all symptoms orsigns of a disease.

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carriere.g. a carrier commonly used in the pharmaceutical industry. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. In some embodimentsof any of the aspects, a pharmaceutically acceptable carrier can be acarrier other than water. In some embodiments of any of the aspects, apharmaceutically acceptable carrier can be a cream, emulsion, gel,liposome, nanoparticle, and/or ointment. In some embodiments of any ofthe aspects, a pharmaceutically acceptable carrier can be an artificialor engineered carrier, e.g., a carrier that the active ingredient wouldnot be found to occur in in nature.

As used herein “combination” refers to a group of two or more substancesfor use together, e.g., for administration to the same subject. The twoor more substances can be present in the same formulation in anymolecular or physical arrangement, e.g, in an admixture, in a solution,in a mixture, in a suspension, in a colloid, in an emulsion. Theformulation can be a homogeneous or heterogenous mixture. In someembodiments of any of the aspects, the two or more substances activecompound(s) can be comprised by the same or different superstructures,e.g., nanoparticles, liposomes, vectors, cells, scaffolds, or the like,and said superstructure is in solution, mixture, admixture, suspensionwith a solvent, carrier, or some of the two or more substances.Alternatively, the two or more substances can be present in two or moreseparate formulations, e.g., in a kit or package comprising multipleformulations in separate containers, to be administered to the samesubject.

A kit is an assemblage of materials or components, including at leastone reagent described herein. The exact nature of the componentsconfigured in the kit depends on its intended purpose. In someembodiments of any of the aspects, a kit includes instructions for use.“Instructions for use” typically include a tangible expressiondescribing the technique to be employed in using the components of thekit, e.g., to treat a subject or for administration to a subject. Stillin accordance with the present invention, “instructions for use” mayinclude a tangible expression describing the preparation of at least onereagent described herein, such as dilution, mixing, or incubationinstructions, and the like, typically for an intended purpose.Optionally, the kit also contains other useful components, such as,measuring tools, diluents, buffers, syringes, pharmaceuticallyacceptable carriers, or other useful paraphernalia as will be readilyrecognized by those of skill in the art.

The materials or components assembled in the kit can be provided to thepractitioner stored in any convenient and suitable ways that preservetheir operability and utility. For example, the components can be indissolved, dehydrated, or lyophilized form; they can be provided atroom, refrigerated or frozen temperatures. The components are typicallycontained in suitable packaging material(s). As employed herein, thephrase “packaging material” refers to one or more physical structuresused to house the contents of the kit, such as inventive compositionsand the like. The packaging material is constructed by well-knownmethods, preferably to provide a sterile, contaminant-free environment.The packaging may also preferably provide an environment that protectsfrom light, humidity, and oxygen. As used herein, the term “package”refers to a suitable solid matrix or material such as glass, plastic,paper, foil, polyester (such as polyethylene terephthalate, or Mylar)and the like, capable of holding the individual kit components. Thus,for example, a package can be a glass vial used to contain suitablequantities of a composition containing a volume of at least one reagentdescribed herein. The packaging material generally has an external labelwhich indicates the contents and/or purpose of the kit and/or itscomponents.

As used herein, the term “nanoparticle” refers to particles that are onthe order of about 1 to 1,000 nanometers in diameter or width. The term“nanoparticle” includes nanospheres; nanorods; nanoshells; andnanoprisms; these nanoparticles may be part of a nanonetwork. The term“nanoparticles” also encompasses liposomes and lipid particles havingthe size of a nanoparticle. Exemplary nanoparticles include lipidnanoparticles or ferritin nanoparticles. Lipid nanoparticles cancomprise multiple componenents, including, e.g., ionizable lipids (suchas MC3, DLin-MC3-DMA, ALC-0315, or SM-102), pegylated lipids (such asPEG2000-C-DMG, PEG2000-DMG, ALC-0159), phospholipids (such as DSPC), andcholesterol.

Exemplary liposomes can comprise, e.g., DSPC, DPPC, DSPG, Cholesterol,hydrogenated soy phosphatidylcholine, soy phosphatidyl choline,methoxypolyethylene glycol (mPEG-DSPE) phosphatidyl choline (PC),phosphatidyl glycerol (PG), distearoylphosphatidylcholine, andcombinations thereof.

As used herein, the term “administering,” refers to the placement of acompound as disclosed herein into a subject by a method or route whichresults in at least partial delivery of the agent at a desired site.Pharmaceutical compositions comprising the compounds disclosed hereincan be administered by any appropriate route which results in aneffective treatment in the subject. In some embodiments, administrationcomprises physical human activity, e.g., an injection, act of ingestion,an act of application, and/or manipulation of a delivery device ormachine. Such activity can be performed, e.g., by a medical professionaland/or the subject being treated.

As used herein, “contacting” refers to any suitable means fordelivering, or exposing, an agent to at least one cell. Exemplarydelivery methods include, but are not limited to, direct delivery tocell culture medium, perfusion, injection, or other delivery method wellknown to one skilled in the art. In some embodiments, contactingcomprises physical human activity, e.g., an injection; an act ofdispensing, mixing, and/or decanting; and/or manipulation of a deliverydevice or machine.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean ±1%.

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

As used herein, the term “specific binding” refers to a chemicalinteraction between two molecules, compounds, cells and/or particleswherein the first entity binds to the second, target entity with greaterspecificity and affinity than it binds to a third entity which is anon-target. In some embodiments, specific binding can refer to anaffinity of the first entity for the second target entity which is atleast 10 times, at least 50 times, at least 100 times, at least 500times, at least 1000 times or greater than the affinity for the thirdnontarget entity. A reagent specific for a given target is one thatexhibits specific binding for that target under the conditions of theassay being utilized.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art to which thisdisclosure belongs. It should be understood that this invention is notlimited to the particular methodology, protocols, and reagents, etc.,described herein and as such can vary. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims. Definitions of common terms in immunology andmolecular biology can be found in The Merck Manual of Diagnosis andTherapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018(ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), TheEncyclopedia of Molecular Cell Biology and Molecular Medicine, publishedby Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8);Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway'sImmunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W.Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's GenesXI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055);Michael Richard Green and Joseph Sambrook, Molecular Cloning: ALaboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., BasicMethods in Molecular Biology, Elsevier Science Publishing, Inc., NewYork, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology:DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); CurrentProtocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), JohnWiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocolsin Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons,Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan,ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe,(eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737),the contents of which are all incorporated by reference herein in theirentireties.

One of skill in the art can readily identify a chemotherapeutic agent ofuse (e.g. see Physicians' Cancer Chemotherapy Drug Manual 2014, EdwardChu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles ofCancer Therapy, Chapter 85 in Harrison's Principles of InternalMedicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era ofMolecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 inAbeloff's Clinical Oncology, 2013 Elsevier; and Fischer D S (ed): TheCancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).

In some embodiments of any of the aspects, the disclosure describedherein does not concern a process for cloning human beings, processesfor modifying the germ line genetic identity of human beings, uses ofhuman embryos for industrial or commercial purposes or processes formodifying the genetic identity of animals which are likely to cause themsuffering without any substantial medical benefit to man or animal, andalso animals resulting from such processes.

In all embodiments where a sample is obtained or has been obtained orprovided, the sample can be sample taken, obtained, or provided viaminimally invasive methods and/or involves only a minor intervention. Insome embodiments of any of the aspects, a sample is taken, obtained, orprovided by one or more of a blood draw or prick, an epidermal or mucusmembrane swab, buccal sampling, saliva sample, a epidermal skin samplingtechnique, and/or collection of a secreted or expelled bodily fluid(e.g., mucus, urine, sweat, etc), fecal sampling, semen/seminal fluidsampling, or clippings (e.g., of hair or nails). In some embodiments ofany of the aspects, the sample comprises, consists of, or consistsessentially of blood (or any fraction or component thereof), serum,urine, mucus, epithelial cells, saliva, buccal cells, a secreted orexpelled bodily fluid, and/or hair or nail clippings.

Other terms are defined herein within the description of the variousaspects of the invention.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

In some embodiments, the present technology may be defined in any of thefollowing numbered paragraphs:

1. A method comprising: determining the expression of at least one geneselected from the group consisting of:

-   -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5,        Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),        E-cadherin (CDH1), ZEB1, AHNAK, miR-424-5p, miR-326, miR424-5p,        miR-27a-3p, miR320b, and miR320d;        in an exosome obtained from a subject.

2. The method of paragraph 1, wherein the expression of at least onegene selected from the group consisting of:

-   -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,        miR-424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b, and        miR320d;        is determined.

3. The method of paragraph 1, wherein the expression of at least onegene selected from the group consisting of:

miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, and miR-375;

is determined.

4. The method of paragraph 1, wherein the expression of at least twogenes selected from the group consisting of:

-   -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, and miR-375;        is determined.

5. The method of paragraph 1, wherein the expression of at least threegenes selected from the group consisting of:

-   -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, and miR-375;        is determined.

6. The method of paragraph 1, wherein the expression of at least fourgenes selected from the group consisting of:

-   -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, and miR-375;        is determined.

7. The method of paragraph 1, wherein the expression of at leastmiR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, and miR-375; isdetermined.

8. The method of paragraph 1, wherein the expression of at leastmiR374a-5p is determined.

9. The method of any of the preceding paragraphs, wherein an increasedlevel of expression of at least one gene selected from:

-   -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5,        Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),        E-cadherin (CDH1), ZEB1, and AHNAK; or        a decreased level of expression of at least one gene selected        from:    -   miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d;        indicates an increased risk of cancer, metastasis, and/or EMT        for the subject, wherein the level of expression is relative to        the level of expression in a exosome obtained from a healthy        non-diabetic subject.

10. The method of any of the preceding paragraphs, further comprising:

-   -   a) i) administering a glucose-controlling medication or obesity        medication and/or        -   ii) administering CT scans at a frequency of higher than 1            CT scan every 6 months, to a subject determined to have an            expression level of at least one gene selected from:            miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,            TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin            (VIM), E-cadherin (CDH1), ZEB1, and AHNAK which is increased            relative to a reference; or an expression level of at least            one gene selected from: miR424-5p, miR-326, miR424-5p,            miR-27a-3p, miR320b and miR320d; which is decreased relative            to a reference.

11. The method of any of the preceding paragraphs, further comprising:

-   -   a) i) administering a glucose-controlling medication or obesity        medication and/or        -   ii) administering CT scans at a frequency of higher than 1            CT scan every 6 months, to a subject determined to have an            expression level of at least one gene selected from:            miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,            TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin            (VIM), E-cadherin (CDH1), ZEB1, and AHNAK which is increased            relative to a reference; or an expression level of at least            one gene selected from: miR424-5p, miR-326, miR424-5p,            miR-27a-3p, miR320b and miR320d; which is decreased relative            to a reference; or    -   b) i) not administering a glucose-controlling medication or        obesity medication and/or        -   ii) administering CT scans at a frequency of no more than 1            CT scan every 6 months, to a subject determined to have an            expression level of at least one gene selected from:            miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,            TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin            (VIM), E-cadherin (CDH1), ZEB1, and AHNAK which is not            increased relative to a reference; or an expression level of            at least one gene selected from: miR424-5p, miR-326,            miR424-5p, miR-27a-3p, miR320b and miR320d; which is not            decreased relative to a reference.

12. A method of treating cancer, comprising:

-   -   a) i) administering a glucose-controlling medication or obesity        medication and/or        -   ii) administering CT scans at a frequency of higher than 1            CT scan every 6 months, to a subject determined to have an            expression level of at least one gene selected from:            miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,            TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin            (VIM), E-cadherin (CDH1), ZEB1, and AHNAK which is increased            relative to a reference; or an expression level of at least            one gene selected from: miR424-5p, miR-326, miR424-5p,            miR-27a-3p, miR320b and miR320d; which is decreased relative            to a reference.

13. A method of treating cancer, comprising:

-   -   a) i) administering a glucose-controlling medication or obesity        medication and/or        -   ii) administering CT scans at a frequency of higher than 1            CT scan every 6 months, to a subject determined to have an            expression level of at least one gene selected from:            miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,            TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin            (VIM), E-cadherin (CDH1), ZEB1, and AHNAK which is increased            relative to a reference; or an expression level of at least            one gene selected from: miR424-5p, miR-326, miR424-5p,            miR-27a-3p, miR320b and miR320d; which is decreased relative            to a reference; or    -   b) i) not administering a glucose-controlling medication or        obesity medication and/or        -   ii) administering CT scans at a frequency of no more than 1            CT scan every 6 months, to a subject determined to have an            expression level of at least one gene selected from:            miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,            TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin            (VIM), E-cadherin (CDH1), ZEB1, and AHNAK which is not            increased relative to a reference; or an expression level of            at least one gene selected from: miR424-5p, miR-326,            miR424-5p, miR-27a-3p, miR320b and miR320d; which is not            decreased relative to a reference.

14. The method of any of the preceding paragraphs, wherein theglucose-controlling medication is selected from the group consisting of:metformin, a sulfonylurea, a glinide, a SGLT2 inhibitor, and insulin.

15. The method of any of the preceding paragraphs, wherein theglucose-controlling medication is metformin.

16. The method of any of the preceding paragraphs, wherein the obesitymedication selected from the group consisting of: orlistat,phentermine-topiramate, naltrexone-bupropion, liraglutide, semagludtide,setmelanotide, phentermine, benzphetamine, diethylpropion, andphendimetrazine.

17. The method of any of the preceding paragraphs, wherein the level ofexpression is the level of mRNA.

18. The method of any of the preceding paragraphs, wherein the exosomeis 30-90 nm in diameter.

19. The method of any of the preceding paragraphs, wherein the exosomeoriginates from a non-tumor tissue.

20. The method of any of the preceding paragraphs, wherein the exosomeis isolated from a non-tumor tissue and/or cells.

21. The method of any of the preceding paragraphs, wherein the non-tumortissue and/or cells is blood, plasma, adipose tissue, adipocytes, orbone.

22. The method of any of the preceding paragraphs, wherein the methodfurther comprises determining the expression level of at least one geneselected from COMP, TSP5, BRD2, BRD3, miR103a, and SOX-2-OT in tumortissue obtained from the subject.

23. The method of any of the preceding paragraphs, wherein the cancer isan epithelial cancer.

24. The method of any of the preceding paragraphs, wherein the cancer isan epithelial adenocarcinoma.

25. The method of any of the preceding paragraphs, wherein the cancer isesophageal cancer, pancreatic cancer, cervical cancer, colorectalcancer, gastric cancer, lung cancer, uterine caner, renal cancer, breastcancer, or prostate cancer.

26. The method of any of the preceding paragraphs, wherein the cancer isbreast and/or prostate cancer.

27. The method of any of the preceding paragraphs, wherein the subjectis diabetic, overweight, and/or obese.

28. The method of any of the preceding paragraphs, wherein the subjectis identified as diabetic when they are determined to have HbA1c of 6.5%or greater, or by fasting glucose or fasting insulin.

29. A method of treating cancer in a subject in need thereof, the methodcomprising administering to the subject exosomes which are:

-   -   a) from a non-diabetic and/or non-obese donor; and/or    -   b) determined to have an level of expression of at least one        gene selected from:        -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,            TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin            (VIM), E-cadherin (CDH1), ZEB1, and AHNAK; which is not            increased; and/or        -   a level of expression of at least one gene selected from:        -   miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and            miR320d; which is not increased, wherein the level of            expression is relative to the level of expression in a            exosome obtained from a healthy non-diabetic subject.

30. A method of treating cancer in a subject in need thereof, the methodcomprising administering to the subject:

-   -   a) an inhibitor of at least one gene selected from:        -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,            TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin            (VIM), E-cadherin (CDH1), ZEB1, and AHNAK; which is not            increased; and/or    -   b) an agonist of at least one gene selected from:        -   miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and            miR320d;

31. The method of paragraph 29 or 30, wherein the subject is onedetermined to have: an increased level of expression of at least onegene selected from:

-   -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5,        Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),        E-cadherin (CDH1), ZEB1, and AHNAK; or        a decreased level of expression of at least one gene selected        from:    -   miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d.

32. The method of any one of paragraphs 29-31, wherein the cancer isepithelial cancer.

33. The method of any one of paragraphs 29-32, wherein the cancer isprostate cancer.

34. The method of any one of paragraphs 29-33, wherein the subject inneed of treatment for cancer is diabetic and/or obese.

35. The method of any one of paragraphs 29-34, whereby EMT is reduced inthe subject.

36. The method of any one of paragraphs 29-35, wherein the subject isfurther administered a BET inhibitor or PROTAC degrader.

In some embodiments, the present technology may be defined in any of thefollowing numbered paragraphs:

1. A method comprising: determining the expression of at least one geneselected from the group consisting of:

-   -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5,        Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),        E-cadherin (CDH1), ZEB1, AHNAK, miR-424-5p, miR-326, miR424-5p,        miR-27a-3p, miR320b, and miR320d;        in an exosome obtained from a subject.

2. The method of paragraph 1, wherein the expression of at least onegene selected from the group consisting of:

-   -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,        miR-424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b, and        miR320d;        is determined.

3. The method of paragraph 1, wherein the expression of at least onegene selected from the group consisting of:

-   -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, and miR-375;        is determined.

4. The method of paragraph 1, wherein the expression of at least twogenes selected from the group consisting of:

-   -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, and miR-375;        is determined.

5. The method of paragraph 1, wherein the expression of at least threegenes selected from the group consisting of:

-   -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, and miR-375;        is determined.

6. The method of paragraph 1, wherein the expression of at least fourgenes selected from the group consisting of:

-   -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, and miR-375;        is determined.

7. The method of paragraph 1, wherein the expression of at leastmiR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, and miR-375; isdetermined.

8. The method of paragraph 1, wherein the expression of at leastmiR374a-5p is determined.

9. The method of paragraph 1, further comprising:

-   -   i) administering a glucose-controlling medication or obesity        medication and/or    -   ii) administering CT scans at a frequency of higher than 1 CT        scan every 6 months, to a subject determined to have an        expression level of at least one gene selected from: miR374a-5p,        miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail        (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),        E-cadherin (CDH1), ZEB1, and AHNAK which is increased relative        to a reference; or an expression level of at least one gene        selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p,        miR320b and miR320d; which is decreased relative to a reference.

10. The method of paragraph 1, further comprising:

-   -   i) administering a glucose-controlling medication or obesity        medication and/or    -   ii) administering CT scans at a frequency of higher than 1 CT        scan every 6 months, to a subject determined to have an        expression level of at least one gene selected from: miR374a-5p,        miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail        (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),        E-cadherin (CDH1), ZEB1, and AHNAK which is increased relative        to a reference; or an expression level of at least one gene        selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p,        miR320b and miR320d; which is decreased relative to a reference;        or    -   i) not administering a glucose-controlling medication or obesity        medication and/or    -   ii) administering CT scans at a frequency of no more than 1 CT        scan every 6 months, to a subject determined to have an        expression level of at least one gene selected from: miR374a-5p,        miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail        (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),        E-cadherin (CDH1), ZEB1, and AHNAK which is not increased        relative to a reference; or an expression level of at least one        gene selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p,        miR320b and miR320d; which is not decreased relative to a        reference.

11. A method of treating cancer, comprising:

-   -   i) administering a glucose-controlling medication or obesity        medication and/or    -   ii) administering CT scans at a frequency of higher than 1 CT        scan every 6 months, to a subject determined to have an        expression level of at least one gene selected from: miR374a-5p,        miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail        (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),        E-cadherin (CDH1), ZEB1, and AHNAK which is increased relative        to a reference; or an expression level of at least one gene        selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p,        miR320b and miR320d; which is decreased relative to a reference.

12. The method of paragraph 11, further comprising:

-   -   i) not administering a glucose-controlling medication or obesity        medication and/or    -   ii) administering CT scans at a frequency of no more than 1 CT        scan every 6 months, to a subject determined to have an        expression level of at least one gene selected from: miR374a-5p,        miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail        (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),        E-cadherin (CDH1), ZEB1, and AHNAK which is not increased        relative to a reference; or an expression level of at least one        gene selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p,        miR320b and miR320d; which is not decreased relative to a        reference.

13. The method of paragraph 11, wherein the glucose-controllingmedication is selected from the group consisting of: metformin, asulfonylurea, a glinide, a SGLT2 inhibitor, and insulin; or the obesitymedication selected from the group consisting of: orlistat,phentermine-topiramate, naltrexone-bupropion, liraglutide, semagludtide,setmelanotide, phentermine, benzphetamine, diethylpropion, andphendimetrazine.

14. The method of paragraph 1, wherein the level of expression is thelevel of mRNA.

15. The method of paragraph 1, wherein the exosome originates from or isisolated from a non-tumor tissue or cells.

16. The method of paragraph 15, wherein the non-tumor tissue or cells isblood, plasma, adipose tissue, adipocytes, or bone.

17. The method of paragraph 11, wherein the cancer is an epithelialcancer.

18. The method of paragraph 17, wherein the cancer is an epithelialadenocarcinoma, esophageal cancer, pancreatic cancer, cervical cancer,colorectal cancer, gastric cancer, lung cancer, uterine caner, renalcancer, breast cancer, or prostate cancer.

19. The method of paragraph 11, wherein the subject is diabetic,overweight, or obese.

20. A method of treating cancer in a subject in need thereof, the methodcomprising administering to the subject exosomes which are:

-   -   a) from a non-diabetic and/or non-obese donor; and/or    -   b) determined to have an level of expression of at least one        gene selected from: miR374a-5p, miR-93-5p, miR-28-3p,        miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist (TWIST1),        Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, and        AHNAK; which is not increased; and/or        -   a level of expression of at least one gene selected from:        -   miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and            miR320d; which is not increased, wherein the level of            expression is relative to the level of expression in a            exosome obtained from a healthy non-diabetic subject.

21. A method of treating cancer in a subject in need thereof, the methodcomprising administering to the subject:

-   -   a) an inhibitor of at least one gene selected from:        -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375,            TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin            (VIM), E-cadherin (CDH1), ZEB1, and AHNAK; which is not            increased; and/or    -   b) an agonist of at least one gene selected from:        -   miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and            miR320d;

22. The method of paragraph 20, wherein the subject is one determined tohave: an increased level of expression of at least one gene selectedfrom:

-   -   miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5,        Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM),        E-cadherin (CDH1), ZEB1, and AHNAK; or        a decreased level of expression of at least one gene selected        from:    -   miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d.

The technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.

EXAMPLES Example 1

Breast and prostate cancer patients who also have chronic inflammatorydiseases, such as Type 2 diabetes, have a higher risk of metastasis thanpatients with the same stage and type of cancer who have normalImmunometabolism. There is a need for new patient treatment paradigmsthat rely on assessment of the patient's immunometabolism state whichthen informs changes to clinical management. Most cancer biomarkers relyon markers derived from or induced by cancer cells. Remarkably, thesystem described herein to assess cancer risk relies on signals fromnon-tumor tissue which are shown to induce dangerous changes in cancercells. Novel diagnostic tools are needed because cancer patientmetabolism, medications and adipocyte or bone health are typically notconsidered in evaluating risk for progression or metastasis of thesecancers. Clinical decision making could be greatly improved for patientsat-risk for cancer progression on account of their metabolicco-morbidities. The >100 million Americans who are diabetic orpre-diabetic at present are insufficiently served the standard of carein breast and prostate medical oncology.

The inventors have identified small extracellular vesicles calledexosomes (about 30 -90 nm in diameter) that originate in non-tumortissue, such as fat or bone, that carry molecular signals that promotemore dangerous (pro-metastatic) changes in breast or prostate cancercell lines, respectively. These factors are also found in the peripheralblood of cancer patients and are used to profile to assist clinicaldecision making about risks for progression and metastasis. Thisinformation can be used to influence a clinician's choices amongtherapeutic options, and help patients understand their risks. Drugsthat improve metabolism and inflammation may reduce this biomarker,which is expected to reduce risk for cancer metastasis, and would informthe patient/clinician decision making over time.

The invention encompasses a method of isolating exosomes and using genesignatures from patient blood, plasma or other non-malignant tissue todetermine patient risk signature for cancer metastasis. The inventionalso includes how such signatures are used to define subsequentpatient-specific therapies. The methodology to assess and profile riskusing exosome plasma biomarkers is applied to profile patients that haveor may be at risk for a variety of epithelial adenocarcinoma solidtumors including but not limited to esophageal cancer, pancreaticcancer, cervical cancer, colorectal cancer, gastric cancer, lung cancer,uterine, renal, breast and prostate cancers. The use of adipocytes froma tissue biopsy to assess and profile cancer risk assessment is alsonovel. The invention specifically includes assessment of cancer EMTmarkers (associated with progression and metastasis) using exosomesisolated from blood or plasma of patients in order to delineatesubsequent therapy and to profile patients for risk of metastasis. Thetool provides a functional measure of progression risk, beyond anassociation of an analyte with a disease state, and can provide a highlypersonalized profile when tested against a standard array of EMT genes(such as commercially available 87-gene arrays).

The approach has the potential to capture data of added value to cancerdisparities populations. For example, a recent paper in JAMA Oncology(PMID: 33475714) draws the important conclusion from a large cohortstudy that Black women in the U.S. are more likely to have a high-riskrecurrence score and to die of axillary node-negative breast cancer thannon-Hispanic White women with comparable recurrence scores. The OncotypeDX Breast Recurrence Score test for a 21-gene signature has lowerprognostic accuracy in Black women, suggesting that personalized assaysused to identify candidates for adjuvant chemotherapy requires modelcalibration in populations with greater racial/ethnic diversity. Thusthis more personalized diagnostic information provided by the presentmethod will improve risk assessment and save lives.

Certain patients, such as diabetic patients, especially benefit from themethods of this invention. Diabetic patients are identified usingstandard methods, including HbA1c which is used as a quick testclinically to determine who is diabetic (6.5% or greater). Fastingglucose and fasting insulin are desirable, and more preferred inclinical studies because then a measure called HOMA (homeostasis modelof insulin resistance) can be calculated. In the general method of theinvention, exosomes are then isolated from blood or plasma, fractionatedas appropriate, and profiled for transcriptional upregulation. In oneexample of the invention, the readout is transcriptional upregulation offour gene sets (AHNAK, SNAI1 SNAI2 and CDH1) which determines metastaticcancer risk.

In another example, the invention is a system for ranking cancerprogression in a diabetic patient diagnosed with a primary breast tumor,comprising isolation of exosomes from blood, plasma or primaryadipocytes of said patient; screening exosomes for a set of genes usingsequencing, RT-PCR or array methods and in some cases relative to acontrol; determining the fold-change of the set of genes; and rankingthe disease progression potential in the patient. One set of genes ofthe invention comprises Snail (SNAI1), Twist (TWIST1), Slug (SNAI2),vimentin (VIM), E-cadherin (CDH1) and ZEB1; a control gene may beβ-actin (ACTB). Patients may be ranked as being high risk, moderate, orlow risk according to the fold change vs. control. In one example of theinvention, high risk is defined as >2.5 fold change in at least 4 of the6 genes vs. control, moderate risk is >1.25 fold but less than 2.5 foldvs. control, and low risk is less than 1.25 fold vs. control.

Example 2

Obesity and metabolic diseases, such as insulin resistance and type 2diabetes (T2D), are associated with metastatic breast cancer inpost-menopausal women. Described herein is the investigation of thecritical cellular and molecular factors behind this link. It was foundthat primary human adipocytes shed extracellular vesicles-specificallyexosomes—that induced the expression of genes associated withepithelial-to-mesenchymal transition (EMT) and cancer stem-like cell(CSC) traits in cocultured breast cancer cell lines. Transcription ofthese genes was further increased in cells exposed to exosomes shed fromT2D patient-derived adipocytes or insulin-resistant adipocytes andrequired the epigenetic reader proteins BRD2 and BRD4 in recipientcells. The thrombospondin family protein TSP5, which is associated withcancer, was more abundant in exosomes from T2D or insulin-resistantadipocytes and partially contributed to EMT in recipient cells.Bioinformatic analysis of breast cancer patient tissue showed thatgreater co-expression of COMP (which encodes TSP5) and BRD2 or BRD3correlated with poorer prognosis, specifically decreased distantmetastasis-free survival. Our findings reveal a mechanism ofexosome-mediated crosstalk between metabolically abnormal adipocytes andbreast cancer cells that may promote tumor aggressiveness in T2Dpatients.

Obesity, insulin resistance and Type 2 diabetes (T2D) are risk factorsfor breast cancer in post-menopausal women (1). The metabolic andinflammatory complications of obesity are implicated in carcinogenesisand progression of estrogen receptor (ER)-positive breast cancer (2).Population studies also establish that obesity-driven T2D associateswith incidence (3), progression and mortality (4) of ER-negative breastcancer. However, the cellular and molecular pathways that mediate breastcancer incidence, progression and metastasis are still not fullydelineated. In sporadic breast cancer, well known genes that controlproliferation, cell cycle, signal transduction, genome stability andother pathways (genes such as MYC, CCND1, ERBB2, TP53 and CDH1)accumulate mutations and contribute in a multistep fashion to expansionof the malignant clone, immune evasion, cell survival, tissue invasionand metastasis. The tumor microenvironment (TME) plays a critical roleto promote cancer progression; however, the adipocytes, endothelialcells, immune infiltrates and other somatic cells of the breasttypically do not harbor any mutations. Thus, DNA mutational databasesare insufficient to understand most TME mechanisms. Co-morbidimmunological or metabolic diseases alter the function of non-mutatedcells systemically and in the breast TME, which creates opportunities toexplore why tumors may progress in one person but not another, despite asimilar mutational landscape in the malignant clones. Herein, theinventors focused on differences in the TME of obesity that might revealnovel mechanisms that promote breast cancer progression.

Adipocytes are by mass the preponderant non-malignant cell type in theTME of breast cancer. Yet, compared to immune infiltrates, adipocytesare disproportionately understudied as modifiers of cancer progression.Adipocytes function as an active endocrine tissue, releasing adipokines,such as interleukin-6, tumor necrosis factor (TNF)-a, leptin andadiponectin (5) that play critical roles in tumor cell proliferation, aswell as matrix metalloproteinases (MMPs) (6) that are important fortumor invasiveness. Adipocytes also release lipid that nearby breasttumor cells adapt for fuel by fatty acid oxidation, becoming moreaggressive upon this metabolic reprogramming (7).

As the obesity epidemic continues to deepen worldwide, understanding thenature and function of adipocyte intercellular communication isincreasing in importance. Extracellular vesicles and a subtype thereofcalled exosomes have gained attention as facilitators of adipocytecrosstalk with nearby tissues, potentially including malignant orpre-malignant breast epithelial cells (2,7). Adipocyte dysfunction is along-appreciated feature of obesity-associated metabolic diseases,including insulin resistance, glucose intolerance, and T2D. Here, theinventors investigated whether the altered secretome of metabolicallydysfunctional adipocytes, including changes to the adipocyte exosomeprofile, may promote breast cancer development and progression. Thepresent investigation focused on novel exosome crosstalk betweenmetabolically abnormal adipocytes and breast cancer cells.

Results

Adipocyte-Tumor Cell Co-Culture as a Microenvironment Model

To investigate the role of adipocytes in breast cancer progression, theinventors first used a transwell system to co-culture the humanER-positive cell line MCF7 with human adipocytes that had beendifferentiated from primary pre-adipocytes surgically obtained fromcancer-free, female patients with or without T2D undergoing bariatricsurgery. It was observed that the expression of key genes that areimportant for epithelial-to-mesenchymal transition (EMT), atranscriptional signature associated with tumor progression (8;including SNAIL, SNAI2, VIM, CDH2 and TWIST]; and MMPs including MMP3and MMP9) were increased upon co-culture (FIG. 1A and data not shown).Fold-changes in EMT gene expression were greater if the adipocytes hadfirst been rendered insulin resistant (IR) by overnight exposure tolow-dose TNFa (9). Ingenuity Pathway Analysis (IPA) of differentiallyexpressed genes (FIG. 1B and data file S2) indicated that IR-convertedadipocytes (FIG. 1C) more strongly induced EMT pathways in co-culturedMCF7 cells than did insulin-sensitive (IS) adipocytes, as compared toMCF7 controls alone (without co-culture). To explore how crosstalkwithin the TME induces such transcriptional signatures and complexcellular phenotypes, the inventors focused on the induction of EMTgenes. This approach permitted exploration of how novel crosstalk in theTME induces transcriptional signatures and more complex cellularphenotypes than just growth, such as morphology, migration and stemness.

The inventors hypothesized that adipocyte-origin exosomes; wereconveying signals to tumor cells. Exosomes were purified from IS or IRadipocyte conditioned media, and used to challenge MCF7 cultures andtest function on a target set of EMT genes (FIG. 1D, 6A). Consistentwith EMT, exosomes attenuated the expression of genes that encodecharacteristic epithelial markers, like E-cadherin (CDH1) (FIG. 6A).Exosomes from undifferentiated, pre-adipocyte controls did not induceEMT genes (FIG. 1D). Several gene expression signatures associated withother important pathways, such as cell cycle or cytokine signaling, wererelatively unperturbed in tumor cells by co-culture with adipocytes(FIG. 6B). Exosome size distributions were similar between exosomes ofIS and IR adipocyte origin (FIG. 6C). Described herein are mechanismsthat depend on adipocyte-origin exosomes. Furthermore, these exosomesconvey more dangerous signals if the adipocytes are insulin resistant orobtained from patients with obesity-associated metabolic complications.

Adipocyte Exosomes Encode Metabolic Status

In post-menopausal women with obesity, adipokines and adipocyte-inducedestrogen production (14), pro-inflammatory, CD68+adipose tissuemacrophages in “crown-like structures” (15), and adipocyte-released freefatty acids (7) have each been implicated in ER+ breast tumorprogression. The association of obesity-driven T2D with elevated riskfor progression of ER-negative breast cancer (3,4), suggests novel THEcrosstalk drives tumor progression independent of tumor hormone status.Thus, the inventors next compared exosomes purified from primaryT2D-derived adipocytes (isolated as pre-adipocyte progenitors frompatients and differentiated ex vivo) to exosomes purified fromadipocytes derived from non-diabetic controls matched by age, sex and 20BMI (FIG. 13). Consistent with exosome results from IR adipocytes,exosomes from T2D adipocytes induced the expression of genes associatedwith pathways linked to breast cancer aggressiveness, such as invasionand migration (FIG. 2A) and EMT (FIG. 7A), compared to non-diabetic (ND)exosomes (data not shown). Immunofluorescence imaging confirmeddifferential regulation of critical EMT proteins (FIG. 2B). Similarresults were obtained for genes and their inferred pathways associatedwith cancer cell sternness (16) (FIG. 2C, 7B). Like exosomes from IS andIR adipocytes, exosome size distributions were similar between exosomesfrom ND and T2D adipocytes (FIG. 7C). Individual transcripts werevalidated by RT-PCR (FIG. 2D and 2E). T2D adipocyte exosomes attenuatedgene expression that is associated with cell death pathways (FIG. 2A-2C)and downregulated those encoding epithelial markers (FIG. 2F). Thus, thedata indicate that patient metabolic status reprograms breast cancercell gene expression—and possibly consequential aggressiveness—throughadipocyte exosomes.

Exosomes from IR Adipocytes Promote Greater EMT and MorphologicalChanges in Cancer Cells, Compared to IS

Characteristic morphological changes also accompany transcription andprotein expression differences in breast tumor cells upon EMT (17),which were measured by image analysis of exosome-treated human cellularmodels (FIG. 3A). The inventors observed increased cellular perimeter,elongation and reduced circularity upon treatment with exosomes derivedfrom T2D adipocytes compared to ND controls (FIG. 3B). Transwellexperiments with MDA-MB-231 cells (18), a model for human triplenegative breast cancer, confirmed that T2D exosomes promote cellmigration (FIGS. 3C and 3D). To identify candidate exosome proteins thatcould account for functional differences between T2D and ND adipocytes,performed proteomics analysis was performed by mass spectrometry (FIG.3E). The full set of peptides identified by mass spectrometry (FIG. 14)was analyzed by IPA. Results showed that pathways associated with tumorcell aggressiveness (motility, invasion and angiogenesis) were increasedin IR exosome payloads compared to IS (FIG. 8), whereas pathwaysassociated with cell death were decreased, consistent with datadescribed above (FIGS. 1 and 2).

It was previously established that critical pathways that promote breastcancer aggressiveness and EMT are controlled by the somatic BET(bromodomain and extraterminal domain) protein family (18,19), althoughonly BRD4, and not BRD2 or BRD3, is required for cellular migration inbreast cancer (18,20) and prostate cancer models (21). Therefore, theBET dependence of exosome induction of selected EMT genes was tested inMCF7 cells. Upon siRNA knockdown of BRD2 and BRD4, but not BRD3, theexosomes purified from ND or T2D adipocytes lost their ability toincrease SNAIL and SNAI2 (FIG. 3F). Thus, exosome signaling to these EMTtarget genes requires the BET proteins BRD2 and BRD4 as essentialeffectors.

The inventors then tested whether the same functional relationships heldfor an entirely different system: 10 murine 4T1 cells (22), a model forhighly metastatic, triple-negative breast cancers (23). Murine 3T3-L1pre-adipocytes differentiated to mature adipocytes (24) were used as thesource of exosomes, then mature adipocytes with murine TNFa were treatedto induce insulin resistance as above. As in the human system, exosomesfrom mouse IR adipocytes induced expression of mesenchymal proteins andreduced epithelial proteins in 4T1 cells, compared to untreated controlor exosomes from IS adipocytes (FIG. 9A). As before, exosomes from IRadipocytes induced EMT genes (FIG. 9B) and greater migration (FIGS.9C-9D) in 4T1 cells than did exosomes from IS adipocytes. As BRD4 is acritical regulator of cellular migration in several human breast cancercell models, including MCF7, SUM149PT and MDA-MB-231 (18), theBRD4-selective PROTAC degrader MZ-1 (25), which ablates prostate cancercell migration (21), was used to inhibit 4T1 cell migration. Asexpected, MZ-1 effectively ablated 4T1 migration provoked by treatmentwith exosomes from IR adipocytes (FIG. 9D).

Compared to Those From Insulin-Sensitive Adipocytes, Exosomes FromInsulin-Resistant Adipocytes Carry More TSP5, Which Induces Several EMTgenes

Both ND and T2D types of exosomes contained proteins of known importancefor the TME. The top differentially represented hit was thrombospondin-5(TSP5, encoded by COMP; FIG. 3E), which is associated with cancerprogression (26,27), suggesting that TSP5 is critical for exosomeeffector function. To test the role of TSP5, the inventors transducedhuman primary pre-adipocytes with lentivirus overexpressing TSP5, thendifferentiated the cells into adipocytes and purified exosomes fromconditioned media. The inventors first confirmed increased expression ofTSP5 mRNA in the adipocytes, and increased protein loading into exosomesusing the exosome marker CD63 as a control (FIG. 4A), then tested EMTgene responses in MCF7 cell readouts. As expected, TSP5-enrichedexosomes significantly increased transcription of ZEB1 and SNAI2 readoutgenes compared to control (FIG. 4B). To prove that exosomes deliver TSP5to MCF7 cells, the inventors loaded synthetic, cationic lipid vesicleswith recombinant human TSP5 and identified them with fluorescentlylabeled ovalbumin. Imunofluorescence showed that TSP5-loaded exosomesindeed delivered their payload to MCF7 cells to induce vimentinexpression (FIG. 4C).

Additionally, exosomes released from TSP5 lentivirus-transduced, primaryadipocytes (ABM-007) induced expected transcriptional changes by EMTarray, whereas control exosomes from the same adipocytes transduced withlentivirus vector control did not (FIG. 4D and data not shown). Here,MMP9 was the top ranked gene. Consistent with previous results, pathwayanalysis TSP5-loaded exosomes increased signaling associated withinvasion, migration and metastasis, and decreased pathways associatedwith cell death (FIG. 4E and data not shown). The invenotrs alsotransfected MDA-MB-231 cells with plasmids for overexpression ofrecombinant, V5 epitope-tagged TSP5 and confirmed by immunofluorescencethat the epithelial marker E-cadherin was decreased (FIG. 10A) and themesenchymal marker vimentin was increased (FIG. 10B) Additionally, theinventors used flow cytometry of CD24 and CD44 surface markers (28) toshow that forced expression of TSP5 shifted non-malignant breastepithelial MCF10A cells toward a more mesenchymal-like (CD44hi/CD24lo)phenotype (FIGS. 11A-11B). These results confirm that forced expressionof TSP5 is necessary and sufficient to induce EMT-like phenotypicchanges in human breast cancer cell models, without ruling out thepossibility that native exosomes released by adipocytes harbor as-yetuncharacterized proteins, enzymes, RNAs or metabolites that provokesimilar shifts (7).

As a negative control, the inventors knocked down Tsp5 in 3T3-L1pre-adipocytes with shRNA and confirmed that knockdown ablated theability of exosomes purified from mature adipocytes to induce severalEMT genes in 4T1 cells (FIG. 11), including SNAI1, NOTCH1, JAG1, ZEB1and MMP3 (FIGS. 12A-12B). Exosomes from the TSPS knockdown adipocytes nolonger induce canonical cancer pathways upregulated by IR exosomes (ILK,ERK/MAPK, HGF and actin cytoskeleton signaling; FIG. 12C). Other IRexosome-induced genes (such as SERPINE1 and MAP1B) were TSP5-independent(FIG. 12B), as were other canonical cancer pathways (such as FAT10cancer signaling, TGFIβ signaling, and STAT3 signaling; FIG. 12C).

Co-Expression of Genes Encoding TSP5 and BET Proteins Associates withDecreased Distant-Metastasis-Free Survival in Breast Cancer Cohorts

Finally, the invenotrs examined distant metastasis-free survival (DMFS)in cohorts of breast cancer patients with high or low expression ofCOMP, the gene encoding TSP5, co-expressed with high or low levels ofeach of the three BET bromodomain genes BRD2, BRD3 or BRD4 (FIG. 5). Asexpected, higher co-expression of COMP and a BET gene was associatedwith reduced DMFS, with the strongest effect for BRD2. Specifically, astatistically significant, 35% increased risk of distant metastasis over25 years was observed for high co-expression of BRD2 and COMP, and a 19%increased risk for high co-expression of BRD3 and COMP. There was atrend for reduced DMFS for high co-expression of BRD4 and COMP, but thedifference did not reach statistical significance for 332 patients pergroup. These findings reaffirm the importance of testing the functionsof all three somatic BET genes (19) in obesity-related cancer, not justBRD4, which is commonly assumed to be the sole important player (29,30).

Discussion

Population studies over twenty years have convincingly implicatedcardiometabolic risk factors in incidence and progression ofobesity-driven cancers, including breast cancers (31-33). Numeroushormones, metabolites, cytokines and tissue structural properties thatchange in concert with obesity, in both humans and animal models, havegained attention as potential mechanisms that link obesity and cancer.However, identification of the most important causal elements has beendifficult. Seeking to leverage ex vivo and in vitro models, manyinvestigators continue to test murine adipocytes against human breastcancer cell lines, in co-culture, conditioned media or organoidexperiments, where species differences may complicate interpretation.Furthermore, the few reports that use human adipocytes often derivepreadipocyte progenitors from normal volunteers and ignore insulinresistance. The inventors determined that human breast cancer cell linesprovide a useful ex vivo readout to assay factors produced by humanprimary adipocytes. The inventors considered the metabolism of theadipocyte as the independent variable.

High-throughput, cellular proliferation assays are widely used fordiscovery of novel biochemical activators or inhibitors, yet thesetechniques yield limited information. The inventors focused instead onpathway analysis of transcriptionally induced EMT genes, which requires3-5 days, rather than simpler, overnight proliferation assays. Thisapproach enabled discovery of novel pathways important for tumorprogression that might be missed with a more convenient assay. Althoughsystemic metabolism of the patient clearly plays a major role in therisk of breast cancer progression (34), the adipocyte contribution tothe local signaling in the TME demands further study.

IPA shows gene regulation pathways associated with tumor cellaggressiveness (invasiveness, migration, angiogenesis) are upregulatedby insulin-resistant cell-derived exosome payloads compared to thosefrom insulin-sensitive cells. Angiogenesis pathways are also coupled tohypoxia in adipose tissue in obesity (37-39), and the increasedexpression of such genes as NOTCH1, SNAI1, SNAI2, SERPINE1, COL1A2, EGFRand those associated with the HIFIα pathway, as shown here, suggest thatinsulin-resistant and T2D-derived adipocyte exosomes may mediatedifferences in the breast TME of patients with metabolic disease,promoting tumor vascularization as the malignant cell clones undergoexpansion. However, breast tumor cell invasiveness and migrationmechanisms appear uniquely to require NOTCH1 and BRD4 signaling (18).

The present results implicate exosome proteins from IR adipocytes intumor aggressiveness associated with EMT and cancer stem cellsignatures. Results are borne out in breast cancer patient cohorts withlong term follow-up (FIG. 5). These conclusions are consistent with anextensive clinical and population literature that has implicated patientmetabolism in risk of breast cancer incidence and progression.Furthermore, adipocyte-origin, circulating exosomes might be suitable asnon-invasive, liquid biopsy biomarkers (40) to assist clinical decisionmaking for breast cancer patients at risk for progression. The presentinsights into exosome communication among adipocytes and tumor models ofdivergent hormone status indicate that patient metabolism influencesprogression risk across breast cancer subtypes, through microenvironmentmechanisms independent of hormone signaling.

Materials and Methods

Cell Lines

Cell lines, including MCF7 (HTB-22), MDA-MB-231 (HTB-26), T47D (HTB-133)and 3T3-L1 (CL-173), were obtained from the American Type CultureCollection (ATCC). Human primary pre-adipocytes were obtained anddifferentiated in Boston University's Adipose Tissue Biology andNutrient Metabolism Core.

Reagents

Unless otherwise specified, chemicals and biochemicals were fromSigma-Aldrich. Differentiation reagents for conversion of murine 3T3-L1fibroblasts to mature adipocytes were dexamethasone (D8893-1MG),3-isobutyl-1-methylxanthine (IBMX, 17018) and insulin (10516). Reagentsused in experiments for transfer of exogenous TSP5 to target cells wererecombinant COMP/TSPS (SRP6457), Pierce™ Protein Transfection Reagent(89850, Thermo Scientific), ovalbumin, and Alexa Fluor™ 647 conjugate(034784, Thermo Scientific). Paraformaldehyde solution (AAJ19943K2,Thermo Scientific) and 4′,6-diamidino-2-phenylindole dihydrochloride(DAPI, FluoroPure grade; D21490, Thermo Scientific) were used to fix andstain the nuclei.

Gene expression analysis by RT-PCR was performed using TaqManTm mastermix (Thermo Fisher, 4369510). The human gene probes were: SNAP(Hs00195591 ml), SNAI2 (Hs00950344 ml), CDH1 (Hs01023895 ml), COMP(Hs00164359 ml), ACTB (Hs00357333 gl). VIM (Hs00958111 ml), ZEB](Hs01566408 ml), TWIST1 (Hs01675818 sl), JAG1 (Hs01070032 ml), NOTCH1(Hs01062014 ml), TGFB1 (Hs00998133 ml), BRD2 (Hs01121986 gl), BRD3(Hs00978972 ml) and BRD4 (Hs04188087 ml).

The mouse gene probes were: Snai1 (Mm00441533 gl), Vim (Mm01333430 ml),Cdhl (Mm0 1247357 ml), Comp (Mm00489490 ml), Twist1 (Mm00442036 ml) andActb (Mm02619580 gl), Zeb1(Mm00495564 ml) and Epcam (Mm00493214 m1).

Antibodies

Antibodies to CD63 (ab216130), TSPS (ab74524), V5 tag (SV5-Pk1)(ab27671) and vimentin (monoclonal ab8978) were purchased from Abcam.Antibodies to E-cadherin (4A2, mouse mAb 14472), vimentin (D21H3, rabbitmAb 5741) and N-cadherin (D4R1H, rabbit mAb 13116) were obtained fromCell Signaling Technology. Filamentous actin (F-actin) was stained usingeither Alexa Fluor™ Phalloidin probes 488nm (A12379), 568nm (A12380) orPlus 647 (A30107) from Thermo Fisher. Mouse or rabbit Alexa Fluor Plussecondary antibodies were purchased from Thermo Fisher and used forimmunofluorescence imaging.

Differentiation of 3T3-L1 Pre-Adipocytes

3T3-L1 pre-adipocytes were cultured in Dulbecco's modified eagle medium(DMEM) containing 4.5 g/L glucose, L-glutamine and sodium pyruvate)supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin,10 μg/mL streptomycin (all from Corning) until confluence, thenincubated in the same medium for an additional 2 days. Differentiationwas induced by addition of 1 μM dexamethasone, 0.5 mM IBMX, and 1.67 μMinsulin for 3 days, at which time the 20 medium was replaced with growthmedium containing 0.41 μM insulin. After differentiation, cells weretreated with 1 nM recombinant mouse TNF-a (Abcam, ab9740) for 24 hoursto induce insulin resistance.

Differentiation of Primary Human Adipocytes

Samples were de-identified and not linked to any protected information.All subjects provided informed consent for participation. The protocolwas approved by Institutional Review Board of Boston University MedicalCenter. To obtain pre-adipocytes, subcutaneous fat tissue was obtainedby BNORC from subjects undergoing bariatric surgery. Adipose stromalcells (ASC) were isolated as previously described (36). Briefly, mincedtissue was treated with collagenase solution (1 mg/mL HBSS) (Type 1,Worthington Biochemical, Lakewood N.J.) at 37° C., shaken at 100 rpm for2 hours. The digested tissue was filtered through a 250 μm mesh(Component Supply, Inc. Smithville Hwy, Sparta, Tenn.). Cells in theflow through were centrifuged at 500×g for 10 min at room temperature.The red blood cells in the cell pellets were lysed (0.154 mM NH4C1, 10mM KH2PO4 and 0.1 mM EDTA, pH 7.3). Then, washed cells were plated usingalpha MEM Media (Gibco Thermo Fisher Scientific, Waltham Mass.) with 10%FBS (Gemini Bio Products, West Sacramento, Calif.), 100 units/mLpenicillin, 10 μg/mL streptomycin (Pen/Strep) (Corning, Corning N.Y.).Differentiation of ASCs to adipocytes was performed in serum-free mediausing chemicals and reagents purchased from Sigma-Aldrich (St. Louis,Mo.) unless otherwise noted. Complete differentiation media (CDM) wasmade in DMEM/F12 (GIBCO) with 25 mM NaHCO3 and Pen/Strep containing 33μM D-(+)-biotin, 17 μM pantothenate, 10 μg/mL transferrin, 100 nMdexamethasone, 100 nM insulin, 1μM rosiglitazone (Calbiochem), 2 nM3,3′,5-triiodo-L-thyronine (T3) and 0.5 mM IBMX. Differentiation wasinitiated two days post-confluence and CDM was replenished every 2-3days. After 7 days, CDM was removed and the cells were fed withmaintenance media: DMEM/F12 (GIBCO) with 25 mM NaHCO3 and Pen/Strepcontaining 33 μM D-(+)-biotin, 17 μM pantothenate, 10 μg/mL transferrin,10 nM dexamethasone and 10 nM insulin.

Negative Control Exosomes From Undifferentiated Adipose Stromal Cells

As a rigorous control to confirm the mature adipocyte origin of thebiologically active exosomes, the inventors used the 2-dimensionalculture of primary ASCs from which adipocytes are differentiated by thestandard cocktail (above) and omitted a key chemical required fordifferentiation: rosiglitazone. In this case, without the consequentactivation of PPARy, mature adipocytes do not form, yet pre-adipocytefibroblasts, endothelial cells and any other ACS cells remainrepresented in the culture. Conditioned media from this pre-adipocyteculture, where differentiation was blocked, was used as the source ofexosomes, which were purified and added to the MCF7 cultures.

Induction of Insulin Resistance

Treatment of mature, fully differentiated, primary adipocytes ex vivowith low concentrations (250 pM) of the pro-inflammatory cytokine tumornecrosis factor alpha (TNFa) is well established to ablateinsulin-sensitive glucose transport and induce phosphorylation of Aktand other molecular changes. Human primary adipocytes were treated with250 pM recombinant human TNFa (Abcam, ab9642), and 3T3-L1 adipocyteswere treated with 1 nM recombinant ouse TNFa (Abcam, ab9740), overnightto induce insulin resistance.

Exosome Isolation

The conditioned media of both ND or T2D adipocytes yielded between8.0×10⁸ and 1×10⁹ exosomes per mL, from which we purified andconcentrated exosomes as follows. Conditioned media (typically 10 mL)was centrifuged at 300×g for 10 min in a 15 mL conical tube to removecells and debris. The supernatant was transferred to a new 15 mL conicaltube and further centrifuged at 16,000×g for 30 min to remove additionaldebris. This supernatant was then transferred to an Amicon Ultra-15(Millipore-Sigma; REF-UFC910008) 100K centrifugal filter and centrifugedat 4,500×g for 15 min to concentrate the material to 1 mL final volume.To precipitate exosomes, 1 mL of this concentrated, conditioned mediawas added to Exo-spin Buffer reagent (Cell Guidance Systems; #EX06) in a2:1 ratio (sample:buffer; v:v) and incubated overnight at 4° C. Thesample was then centrifuged at 16,000×g for 1 hour at 4° C. to pelletthe exosomes. The supernatant was discarded and the pellet wasre-suspended in 100 μL PBS (Cell Guidance System; LOT0619; EX-P10) forfurther separation of exosomes from other exosomes using Exo-spincolumns. These spin columns work on the principle of size exclusionchromatography with separation of particles based on diameter. Thecolumns are packed with spherical beads, with a 30 nm pore size. Thespace between the pores is such that the eluate contains exosomes with asize range of 30 nm-200 nm. The Exo-spin (Cell Guidance System; LOT0720)column was equilibrated for 15 min at room temperature before use. Thecolumn was calibrated twice with 250 μL PBS and centrifuged at 50×g for10 sec. Then, 100 μL of re-suspended total exosome sample was added tothe top of the column, which was then centrifuged 20 at 50×g for 60 secand the flow through was discarded. The column was then transferred to anew 1.5 mL centrifuge tube, whereupon 200 μL PBS was applied to thecolumn, which was then centrifuged at 50×g for 60 sec to elute 200 μL offinal, pure exosomes for downstream functional assays.

Exosomes were also purified using a Total isolation kit (Thermo FisherScientific) and purified by size exclusion chromatography on a qEVOriginal column, using an Automatic Fraction Collector (AFC, serialnumber: V1-0395, IZON) and identified for similar functional outcomes indownstream assays. The size distribution and concentration of exosomeswere always determined before each biological experiment using aNanoSight NS300 system (Malvern Panalytical). Day-to-day variation inexosome yields across adipocyte cultures was <10%, and TSP5 knockdown oroverexpression adipocytes yielded exosome numbers within this range. Foreach experiment, exosome counts were always normalized after NanoSightquantitation such that equal numbers were added to cell wells in eachexperiment and across 10 replicates.

Glucose Transport Assay

To measure glucose uptake in cells, the Glucose Uptake-G1o™ Assay(J1341, Promega) was used as outlined in the manufacturer's protocol.This is a plate-based, bioluminescent method for measuring glucoseuptake by living cells, based on the detection of2-deoxyglucose-6-phosphate (2DG6P).

Cellular Symmetry Analysis

ImageJ was used to analyze cell morphological parameters. Perimeter wasdefined as the length of the outside boundary of a cell. Circularity wascalculated as 4π×area/perimeter² wherein a value of 1.0 indicates aperfect circle. As the value approaches 0.0, the parameter indicates anincreasingly elongated shape. Cell elongation was measured as aspectratio:major axis/minor axis.

Exogenous TSP5 Transfer to Cells

Recombinant TSP5 (Sigma-Aldrich, SRP6457) was loaded into target cellsusing Pierce Protein Transfection Reagent Kit (Thermo Scientific,89850). Reagent protein complexes attach to negatively charged cellsurfaces, and either can directly fuse with the membrane to deliver thecaptured protein into the cell or undergo endocytosis and then fuse withthe endosome, to release the captured protein into the cytoplasm. ThePierce™ Reagent was vortexed for 10 to 20 seconds at top speed beforeeach use. For a 6-well plate or 33 mm dishes, 10 μL of reagent waspipetted. The solvent was evaporated by placing the microcentrifugetubes containing the reagent under a laminar flow hood for 2 hours. Thedried reagent was hydrated with the diluted solution of recombinantprotein and carrier (TSP5, 100 ng+AlexaFluor-647-labelled ovalbumin, 5μg) in 50 to 100 μL PBS. Then, the solution was mixed briefly bypipetting up and down 3 to 5 times, incubated at room temperature for 5min and vortexed for 3 to 5 seconds at low to medium speed. Serum-freemedium was added to the reagent/protein complex to bring the finaldelivery 15 volume up to 1 mL. The final delivery mix was transferredonto PBS-washed cells, incubated for 3 to 4 hours at 37° C., then onevolume of 20% serum-containing medium was added directly to the well ordish to quench the reaction.

TSP5 Knockdown and Overexpression

All lentiviral particles were obtained from Sigma-Aldrich. To depleteTSP5, human primary pre-adipocytes (from subject ID: ABM- 007) weretransduced with pLKO.1-COMP shRNA (TRCN0000056075) and non-targetcontrol (SHC001V) lentiviral particles. To overexpress TSP5, humanprimary pre-adipocytes were transduced with pLX 317-COMP(TRCN0000470297) and control vector (ORFBFPV) lentiviral particles.Human primary pre-adipocytes were selected using growth media containingpuromycin (0.5 μg/mL). To knockdown Tsp5 in 3T3-L1 mouse pre-adipocytes,mouse-specific pLKO.1-COMP shRNA (TRCN0000066166) and non-target control(SHCOO1V) lentiviral particles were used. Cells were selected usinggrowth media containing puromycin (1 μg/mL). After selection in eachcase, the transduced pre-adipocytes were differentiated to matureadipocytes as described above.

Knockdown by siRNA Transfection

Cells were transfected with 25 nmol/L of listed siRNAs for 72 hours withLipofectamine™ RNAiMAX Transfection Reagent (13778075, Thermo Fisher).Human Bromodomain and ExtraTerminal (BET) and non-targeting (scramble)SMARTpool siRNAs were purchased from Dharmacon. The pools in each casewere comprised of five independent siRNAs. Catalog numbers are asfollows: siBRD2 (L-004935-00-0005), siBRD3 (L-004936-00-0005) and siBRD4(L-004937-00-0005).

Immunofluorescence (IF) Imaging

For IF imaging 100K cells were cultured in 27 mm glass bottom dishes(Thermo Fisher, 150682). For staining, cells were washed with DPBSincluding MgC12 and CaC12, then fixed with 4% paraformaldehyde (PFA) for10 min. Then, cell membranes were permeabilized with Triton-X 100, 0.5%for 15 min. Cellular binding sites were blocked with bovine serumalbumin (BSA), 2% (w:v) for 45 min. Then fixed, permeabilized andblocked cells were stained with primary antibodies (1:100 dilution, v:v)for 2 hours, washed 5 times, then stained with IF conjugated secondaryantibodies (1:200 dilution, v:v) and phalloidin (1:1000 dilution, v:v)and DAPI counterstain. Finally, images were captured using a NikonDeconvolution Wide-Field Epifluorescence System at the Boston UniversityCellular Imaging Core. Images were analyzed using ImageJ, withdifferences in means evaluated by comparison of a minimum of 25individual, representative cells for each condition. Images wereassembled in Adobe Photoshop Version 21.1.2. IF stain of proteins withprimarily plasma membrane localization (such as E-cadherin) wasquantified by selecting the plasma membrane and comparing across samplesusing the same method. Other proteins that have cytoplasmic or cell-widedistribution were quantified by selecting the whole cell. For TSPSexosome delivery experiments (FIG. 4), it was found that the extent ofstaining of AlexaFluor 647-ovalbumin carrier protein was not changed bythe presence or absence of TSPS, from which it was concluded that TSPSdoes not alter the ability of exosomes to bind to or merge with thetarget cells

Proteomics Analysis (Nanospray LC-MS/MS Analysis)

Exosome proteomics analysis was performed by Poochon Scientific LLC(Frederick, Md.) using liquid chromatography with tandem massspectrometry (LC-MS/MS). The protein of the exosome samples wasdenatured by 1% SDS and heated at 95° C., followed by digestion withtrypsin (Pierce Trypsin Protease, MS grade; #90057). In brief, thedenatured protein was reduced with dithiothreitol at 56° C. for 45 min,followed by alkylation with iodoacetamide for 30 min at room temperaturein the dark. Alkylated proteins were then precipitated by 80% acetone,followed by trypsin digestion at 37° C. for 16 hours. The digestedpeptide mixture was then concentrated and desalted using C18 Zip-tip(ZTC18S960, Millipore). Reconstituted, desalted peptides were dissolvedin 20 μL of 0.1% formic acid (Formic Acid Optima LC/MS (A11-50), FisherScientific) in LC-MS/MS grade water. Peptides (12 μL) were analyzed by110 min LC-MS/MS run.

The LC-MS/MS analysis of samples was performed using an OrbitrapExploris 240 Mass Spectrometer and a Dionex UltiMate 3000 RSLCnanoSystem (both from Thermo Scientific). The Orbitrap Exploris 240instrument was operated in the data dependent mode to automaticallyswitch between full scan MS and MS/MS acquisition. Peptide mixture fromeach sample was loaded onto a peptide trap cartridge at a flow rate of 5μL/min. The trapped peptides were eluted onto a reversed-phase EasySprayC18 column (Thermo) using a linear gradient of acetonitrile (3-36%) in0.1% formic acid. The elution duration was 110 min at a flow rate of 0.3μL/min. Eluted peptides from the EasySpray column were ionized andsprayed into the mass spectrometer, using a Nano-EasySpray Ion Source(Thermo) under the following settings: spray voltage, 1.6 kV, capillarytemperature, 275° C. The 15 most intense multiply charged ions (z≥2)were sequentially isolated and fragmented in the octopole collision cellby higher-energy collisional dissociation (HCD) using normalized HCDcollision energy 30 with an AGC target 1×105 and a maximum injectiontime of 200 ms at 17,500 resolution. The isolation window was set to 2.The dynamic exclusion was set to 20 s. Charge state screening wasenabled to reject unassigned and 1+ and 7+ or higher charge state ions.

The raw data file acquired from each sample was searched againstUniProtKB human protein sequences database (20,547 entries, downloadedon Apr. 20, 2020) and target protein sequences were searched using theProteome Discoverer 2.4 software (Thermo) based on the SEQUESTalgorithm. Carbamidomethylation (+57.021 Da) of cysteines was fixedmodification, and Oxidation Met and Deamidation Q/N-deamidated (+0.98402Da) were set as dynamic modifications. The minimum peptide length wasspecified to be five amino acids. The precursor mass tolerance was setto 15 ppm, whereas fragment mass tolerance was set to 0.05 Da. Themaximum false peptide discovery rate was specified as 0.01. Theresulting Proteome Discoverer Report contains all assembled proteinswith peptides sequences and peptide spectrum match counts (PSM#).

PCR Array

RNA was isolated from cells using RNeasy Plus Mini Kit (74136, Qiagen).From each sample, 1μg of RNA was used to prepare 20 μL cDNA usingQuantiTect Reverse Transcription Kit (Qiagen, 205313). Human RT2Profiler™ PCR Array, including Epithelial to Mesenchymal Transition(EMT) (PAHS-090Z) and Cancer Stem Cell (PAHS-176ZC) arrays, werepurchased from Qiagen. Each cDNA sample (102 μL comprised of 1 μg RNA)was mixed with 1,350 μL RT2 SYBR Green ROX qPCR Mastermix (Qiagen,330522) and 1,248 μL RNAse-free water. A total volume of 2,700 μL wasmixed by vortexing and transferred to a reservoir. Then, an 8 channelpipettor was used to take aliquots of reaction mix from the reservoir,to distribute 25 μL into each well of the array. PCR reactions wereperformed and results were analyzed using a 7500 Fast Real-Time PCRinstrument.

Gene Expression Analysis

All Ct values of the genes were normalized to the respective ACTB gene(delta Ct). Then the ΔCt of each gene was subtracted from the controlgene ΔCt (delta.delta Ct). For the control groups, delta.delta Ct wascalculated using this formula: ΔΔCt=ΔCt (C1 or C2 or C3)—ΔCt (Controlaverage). Then, fold-change was calculated using 2∧−ΔΔCt, (2 to thepower of negative ΔΔCt). Next, the Z score was calculated based on thisformula: Z score=(x-mean)/SD, where X is the fold-change. Hierarchicalclustering analyses were performed using BioVinci Software (Bioturing,San Diego, Calif., USA). Heatmaps were generated by applying clusteringon rows (either: genes, pathways or diseases) using the Euclideandistance metrics and complete linkage criterion.

Ingenuity Pathway Analysis

To predict disease and function, and downstream pathways, data wereanalyzed through the use of Ingenuity Pathway Analysis, IPA (QIAGENInc.). Available on the world wide web atqiagenbioinformatics.com/products/ingenuity-pathway-analysis). Data wereuploaded to an IPA account through software. Core analysis wasconducted, then comparison analysis was performed on all the conditionsof each experiment, including replicates. The p-value cutoff (log10) wasset to 1.3, which corresponds top-value of 0.05. The measurementactivation Z-score range was −3.5 to +3.5. After running each IPAanalysis, a representative heatmap of proteomics signatures wasgenerated.

Flow Cytometry Analysis

Single cell suspensions were washed after collection and stained inice-cold Ca+2/Mg+2.-free PBS with a viability dye (Zombie NIR,BioLegend) for 20 min at 4° C. in the dark. Cell suspensions were thenwashed twice with ice-cold flow cytometry buffer (Ca+2/Mg+2.-free PBS,supplemented with 2% FBS and 2 mM EDTA). Cell suspensions were thenstained extracellularly with the appropriate antibodies for 25 min at 4°C., in the dark. CD324 (E-cadherin, clone 67A4) was conjugated toPerCP/Cy5.5 (BioLegend), CD44 (clone BJ18) was conjugated to APC(BioLegend) and CD24 (clone ML5) was conjugated to FITC (BDBiosciences). All cell suspensions were washed twice in ice-cold flowcytometry buffer prior to analysis. Unstained cells and single-stainedcontrols were used to calculate flow cytometry compensation. Dataacquisition (typically 1 million events) was performed on a BD LSRIIinstrument at the Boston University Flow Cytometry Core Facility. Dataanalysis was carried out using FlowJo Software (version 10.6.1, TreeStar).

Migration Assay

MDA-MB-231 cells were cultured in DMEM media (25 mM glucose+10% FBS+1%antibiotic supplementation) for 3 days with adipocyte-derived exosomesfrom non-diabetic patients (ND group), Type 2 Diabetic patients (T2Dgroup) and fresh media (exosome control) or PBS (control) for threedays. Cells were then switched to serum-free media for three hours andsubsequently plated in 24-well, 8-micron pore size Transwell plates(Thermo Fisher) for 6 hours.

The cells were plated in the upper well of the transwell inserts withserum free media and the bottom well was filled with DMEM complete mediato serve as a chemoattractant. Cells that stayed in the upper side ofthe membrane and did not migrate by the end of the assay were removedwith a cotton swab, and the cells that migrated were fixed with ice-coldmethanol for 5 minutes at −20° C. After fixation, cells were thenstained with 1% crystal violet (v:v) in 2% ethanol for 10 minutes atroom temperature. Images were captured by an EVOS XL Core digitalinverted microscope. The percentage of migration and invasion wasdetermined by first calculating the sum of the area of totalmigrated/invaded cells on the entire membrane with ImageJ software(National Institutes of Health, Bethesda, Md.), and then converted torelative percent migration/invasion by comparing each condition to thecontrol condition.

Kaplan-Meier Analysis

To investigate the correlation between distant metastasis-free survival(DMFS) and BRD2, BRD3, BRD4 and COMP expression, the inventors uilizeddata from 1803 (BRD2 and BRD3) and 664 (BRD4) patients from the TCGArepository (www.kmplot.com; list reference shown below). The normalizedexpression values of the RNAseq ID 214911 (BRD2), 212547 (BRD3), 226054(BRD4) and 205713 (COMP) were used. For each gene, the mean expressionwas selected and cutoff values were determined by median expression. Toassess the potential additive effects of the genes, samples with “low”BRD2, BRD3 or BRD4 expression and “low” COMP expression were combinedinto separate, individual cohorts and were compared to cohorts with“high” BRD2, BRD3 or BRD4 and “high” COMP expression. Kaplan-Meier plotswere drawn by comparing the “low” and “high” cohorts to estimate theduration of survival. Hazard Ratios (HR) and p values are shown fordistant metastasis-free survival (DMFS) (37).

Statistical Analysis

Statistical analyses for PCR experiments and in vitro cell cultureassays were typically performed using unpaired, two-tailed Student'st-test. Biological and biochemical replicates were performed in at leasttriplicate and data are presented as means. Error bars representstandard error of measurement. Cellular morphology measurements wereperformed with at least 25 representative cells at the samemagnification, in areas of equal culture density. The following symbolswere used to indicate significant differences: ns P>0.05, * P<0.05, **P<0.01, *** P 20<0.005, and **** P<0.001.

Biohazards

All the experiments executed for this study were conducted in accordancewith NIH guidelines, under the oversight of the Boston UniversityInstitutional Biosafety Committee and with approval. Universalprecautions were always observed for human primary tissue or cell lines.

References

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Example 3: Novel plasma exosome biomarkers for prostate cancerprogression in co-morbid metabolic disease

Comorbid Type-2 diabetes (T2D), a metabolic complication of obesity,associates with worse cancer outcomes for prostate, breast, head andneck, colorectal and several other solid tumors. However, the molecularmechanisms remain poorly understood. Exosomes as carriers of miRNAs inblood encode the metabolic status of the originating tissues and delivertheir cargo to target tissues. The inventors hypothesized that T2Dplasma exosomes induce epithelial-mesenchymal transition (EMT) andimmune checkpoints in prostate cancer cells. It is shown herein thatplasma exosomes from subjects with T2D induce EMT features in prostatecancer cells and upregulate the checkpoint genes CD274 and CD155. It isproven herein that specific exosomal miRNAs abundant in T2D plasma(miR374a-5p, miR-93-5p, miR-424-5p) are delivered to tumor cells andregulate these target genes. This builds on the data above showing BRD4controls migration and dissemination of castration-resistant prostatecancer and transcription of key EMT genes, to show that T2D exosomesdrive EMT and immune ligand expression through BRD4. The resultsindicate novel, non-invasive approaches to evaluate prostate cancerprogression risk in patients with comorbid T2D.

Introduction

The incidence of obesity-driven diabetes continues to increaseworldwide, and parallel to this trend, an increase in incidence ofseveral obesity-related cancers has been reported [1]. Already in theU.S., for example, >100 million adults have been diagnosed with diabetesor pre-diabetes [2], about 90% as Type 2 diabetes (T2D) [3], a frequentmetabolic complication of obesity, emphasizing the scale of the publichealth challenge. The severity of comorbid T2D also predicts worsecancer outcomes; associations are well established for breast cancer[4], head and neck cancer [5], colorectal cancer [6] and several othersolid tumors. In prostate [7, 8] and other cancers [9], increasedprevalence of comorbidity also correlates with advanced age [10] andworse outcomes. Yet the cellular and molecular mechanisms that explainthese associations remain poorly understood. Comorbidity isinsufficiently considered in clinical decision making. These knowledgegaps are important for medically underserved patient populations, whereobesity and T2D are prevalent [11, 12], often in association with fooddeserts and an obesogenic built environment [13]. There is urgent needfor novel, robust biomarkers to evaluate risks for cancer progressionand assist clinical decision making for such underserved patients [14,15].

New work in molecular endocrinology is revealing that cancer patientswith comorbid chronic obesity and/or metabolic complications haveadverse signaling in the adipose microenvironments of breast [16-19] orprostate tumors [20-22], compared to patients with the same type andstage of cancer, who are otherwise metabolically normal. In prostatecancer, obesity and its metabolic complications have been studiedintensively to identify serological and histological data that wouldhelp identify cases at high risk for failure of androgen deprivationtherapy (ADT), progression and metastasis in patients with theseco-morbid conditions.

Metabolic biomarkers that include elevated insulin-like growth factor-1,insulin and C-peptide [23, 24], leptin, glucose [25, 26],pro-inflammatory cytokines, lipid profiles [27] and androgen levels haveall been associated with worse outcomes [28, 29]. Metabolic status isalso a concern because ADT is known to induce insulin resistance [30].However, clinical trials have not yet established which serologicalmarkers of metabolism are most informative for the wide range ofprostate cancer patients on various ADT and metabolic medications, andat which stages of disease progression specific markers would havegreatest value. Innovative directions would be helpful.

The inventors took as a starting point the clinical observation thatpoor control of T2D in prostate cancer associates with rapid emergenceof resistance to the anti-androgens abiraterone acetate andenzalutamide, compared to prostate cancer patients with well controlledblood glucose [26]. Noninvasive biomarkers have been explored fordiagnostic, prognostic and therapeutic utility, including most recentlycirculating tumor DNA [31] and microRNAs (miRNA) [32]. In particular,miRNA biomarkers have gained attention because, unlike other nucleicacid biomarkers, these factors may be functional in prostate cancer[33]. Upon delivery to target tissues, miRNAs are capable ofreprogramming cell metabolism and fate to affect the course ofprogression, metastasis and therapeutic responses. Deeper understandingof miRNA mechanism and gene targets may suggest novel therapeutics orprognostic biomarkers [34] to understand progression risks. Theinventors investigated whether circulating miRNAs might differ betweenT2D and non-diabetic (ND) subjects.

Significant evidence implicates exosomes as carriers of miRNAs in blood[35], saliva [36] or other body fluids that can be sampled noninvasivelyfor biomarker assessment in cancer. Several studies have investigatedblood miRNAs derived from tumors as an approach to evaluate cancerdiagnosis [37, 38], prognosis [39] and recurrence [40]. However, theinventors took a converse approach, and investigated plasma exosomalmiRNAs as biomarkers of co-morbid T2D that instead might have functionalimpact on prostate cancer progression. The inventors' rationale is thatobesity-driven metabolic disease has long been studied in prostatecancer incidence [41, 42], progression [43] and prostate cancer-specificmortality [44], although diabetes has been shown to be protective insome prostate cancer studies [45, 46]. Despite intensive investigation,the mechanisms and clinical variables most strongly associated withincidence, progression, distant metastasis and mortality [41, 47], andthe impact of metabolic medications [48], have not been settled. Theinventors considered that plasma exosomes in subjects withobesity-driven T2D might be leveraged to assess risk of progression andtreatment resistance in prostate cancer, and might have functionalsignificance to understand mechanisms of tumor progression.

As shown above herein, the metabolic status of mature adipocytesdetermines the payload of released exosomes. Adipocytes that have beenrendered insulin resistant by exposure to pro-inflammatory cytokines, orthat were isolated from adipose tissue of adult subjects with T2D,release exosomes that drive increased migration, invasiveness,epithelial-to-mesenchymal transition (EMT), gene expression associatedwith cancer stem-like cell formation and aggressive, pro-metastaticbehavior in breast cancer cell models, compared to adipocytes that areinsulin sensitive or isolated from adipose tissue of ND subjects [49].The inventors built on these observations and hypothesized that similarphenotypes would be observed for plasma exosomes from T2D subjectscompared to ND controls, using prostate cancer cell lines as a readout.It is described herein that exosomes from peripheral blood plasma ofsubjects with T2D induce EMT in DU145 cells, a model forhormone-refractory and aggressive prostate cancer.

Interestingly, exosomes purified from the plasma of ND subjectssuppressed transcription of classical EMT genes in the same model,indicating that ND exosomes may harbor cancer chemopreventive miRNAs. Inthe same system it ws found that that T2D exosomes also induceexpression of the immune checkpoint gene CD274, which encodes the immunecheckpoint protein PD-L1. The somatic members of the Bromodomain andExtraTerminal (BET) family of proteins (BRD2, BRD3, BRD4) are positiveco-regulators of the PD-1/PD-L1 axis in triple negative breast cancermodels [50]. BRD4 regulates migration and dissemination ofcastration-resistant prostate cancer and transcription of key EMT genes[51, 52]. Using the pan-BET inhibitor JQ1 and the BRD4- selective PROTACdegrader MZ-1, it is further demonstrated here that BRD4 is a criticaleffector for plasma exosome-driven prostate cancer aggressiveness, andfunctionally couples EMT and immune checkpoint gene expression inprostate cancer. These results establish a deeper, clinicaltranslational investigation of plasma exosomes as functionally criticaldrivers of prostate tumor progression in patients with comorbid T2D, andas biomarkers that are both robust and non-invasive.

Materials and Methods

Cell Lines and Reagents

Cell lines were previously described [51, 52]. The DU145 prostate cancercell line was cultured in RPMI-1640 medium (Gibco). All culture mediawere supplemented with 10% fetal bovine serum (FBS, Corning) and 1%antibiotics (penicillin/streptomycin, Gibco). As positive controls forinduction of EMT genes or PD-L1, we treated the cells with transforminggrowth factor (TGF)-β or interferon (IFN)-γ (5 ng/mL), respectively, aspreviously published [53]. Paraformaldehyde solution (AAJ19943K2, ThermoScientific) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI;FluoroPure grade; D21490, Thermo Scientific) were used to fix and stainthe nuclei. Recombinant Human Interferon gamma protein (Active)(ab259377), and Recombinant human TGF beta 1 protein (ab50036) werepurchased from Abcam.

RNA Staining and Immunofluorescence Imaging

Exosomal RNA was stained using SYTO™ RNASelect™ from Thermofisher (catnumber: S32703) according to the manufacturer's protocol. Alexa Fluor™568 Phalloidin (Thermofisher, A12380) was used to stain the actinfibers. Cellular nuclei were stained by DAPI, FluoroPure™ grade(Thermofisher, D21490).

qRT-PCR

Procedures were as previously described [51, 52]. Briefly, total RNA wasextracted from tumor cells using an RNAeasy Kit (Qiagen). Reversetranscription reactions were performed with 1 μg of total RNA with theQuantiTect Reverse Transcription kit (Qiagen). Gene expression wasmeasured using TaqMan™ master mix (Thermofisher, 4369510) and human geneprobes as follows: SNAI (Hs00195591_m1), SNA2 (Hs00950344_m1), CDH1(Hs01023895_m1), ACTB (Hs00357333_g1), CD274 (encodes PD-L1,Hs01125301_m1), CD155 (encodes PVR or TIGIT ligand, Hs00197846_m1), VIM(Hs00958111_m1), TGFB1 (Hs00998133_m1), and AHNAK (Hs01102463_m1).

PCR Array

RNA was isolated using QuantiTect Reverse Transcription Kit (Qiagen),and 1 μg of each sample was used to prepare 20 μL cDNA using RNeasy PlusMini Kit (Qiagen). Human RT² Profiler™ PCR Array, including Epithelialto Mesenchymal Transition (EMT) genes (PAHS-090Z), Cancer Stem Cellgenes (PAHS-176ZC), and miRNA Array were purchased from Qiagen. Reversetranscription reactions were performed with RT² SYBR Green ROX qPCRMastermix (Qiagen). Exosomal microRNAs were profiled using HumanSerum/Plasma miRCURY LNA miRNA PCR array (Qiagen, #YAHS-106Y, PlateFormat: 2×96-well).

Gene Expression Analysis

All Ct values of the genes were normalized to the respective ACTB gene(ΔCt). Then ΔCt of each gene was subtracted from the control gene ΔCt(Δ.ΔCt). For the control groups Δ.ΔCt was calculated using this formula:ΔΔCt=ΔCt (C1 or C2 or C3)−ΔCt (control average). Then, fold change wascalculated using 2∧−ΔΔCt, (2 to the power of negative ΔΔCt).

Next, the Z score was calculated based on this formula: Zscore=(x-mean)/SD, in which X is the fold change. Bio Vinci software(San Diego, Calif.) was used to cluster the genes. In order to predictdisease and function, data were analyzed through the use of IngenuityPathway Analysis, IPA (QIAGEN Inc., available on the world wide web atqiagenbioinformatics.com/products/ingenuity-pathway-analysis).

Exosome Isolation and Characterization

Patient whole blood was obtained commercially from Research BloodComponents, LLC (Watertown, Mass.) on ice pack, centrifuged (16k rpm, 4°C., 30′) to separate plasma, which was clarified again by centrifugationand filtration (0.2 μm) to remove large vesicles or apoptotic bodies.Exosomes were purified by size exclusion using qEV columns by automaticfractionation collector. The columns were packed with spherical beads(35 nm pore size) such that the spaces between the pores fractionateexosomes with size range of 35 nm-150 nm. The exosomes were eluted usingPBS with 1 mM EDTA and stored at 4° C. for exosome size distribution andconcentration measurements using a NanoSight NS300 system. Exosomalpreparations underwent quality control analysis as previously published[49]. T2D and ND plasma origin exosomes were normalized to 10⁹ particlesadded per cell culture well.

MicroRNA Profiling of Plasma Exosomes

Exosomal RNA was isolated using exoRNeasy Midi Kit (Qiagen, 77144), FormiRNA-specific first-strand cDNA synthesis, miRCURY LNA RT Kit (Qiagen,339340) was used. Then, the PCR array analysis was conducted usingmiRCURY LNA SYBR Green PCR Kit (Qiagen, 339346) and Human Serum/PlasmamiRCURY LNA miRNA PCR array (Qiagen, Catalog Number: YAHS-106Y, PlateFormat: 2×96-Well) to detect plasma exosomes.

microRNA Delivery to DU145 Cells

Differentially expressed microRNAs in T2D exosomes were transfectedusing Lipofectamine RNAiMAX Transfection Reagent (13778150, Invitrogen).The human homologs of the synthesized microRNA mimics were purchasedfrom Thermofisher. The miRBase Accession numbers are as follows:hsa-miR-374a-5p (MIMAT0000727), hsa-miR-93-5p (MIMAT0000093), andhsa-miR-424-5p (MIMAT0001341). After transfection, cells were incubatedfor 48 hr at 37° C., whereupon cellular total RNA was isolated andexpression of SNAI1, PDL1, CDH1 and ACTB was analyzed in transfectedcells.

Flow Cytometry Analysis

Single-cell suspensions were washed after collection and stained inice-cold Ca⁺²/Mg⁺²-free PBS with a viability dye (Zombie NIR, BioLegend)for 20 min at 4° C. in the dark. Cell suspensions were then washed twicewith ice-cold flow cytometry buffer (Ca⁺²/Mg⁺²-free PBS, supplementedwith 2% FBS and 2 mM EDTA). Cell suspensions were then stained for PD-L1(PE-conjugated, BD Biosciences). All cell suspensions were washed twicein ice cold flow cytometry buffer before analysis. Unstained cells andsingle-stained controls were used to calculate flow cytometrycompensation. Data acquisition (typically 1 million events) wasperformed on a BD LSRII instrument at the Boston University FlowCytometry Core Facility. Data analysis was carried out using FlowJoSoftware (version 10.6.1, Tree Star).

RNA Sequencing

Total RNA was isolated from DU145 cells that were untreated controls orwere treated with exosomes isolated from the plasma of three ND subjectsor three T2D subjects. Each experimental group was represented inbiological triplicate. RNA sequencing workflow was conducted by BostonUniversity School of Medicine Microarray and Sequencing Core. FASTQfiles were aligned to human genome build hg38 using STAR [54], (version2.6.0c). Ensembl-Gene-level counts for non-mitochondrial genes weregenerated using featureCounts (Subread package, version 1.6.2) andEnsembl annotation build 100 (uniquely aligned proper pairs, samestrand). Separately, SAMtools (version 1.9) was used to count readsaligning in proper pairs at least once to either strand of themitochondrial chromosome (chrM) or to the sense or antisense strands ofEnsembl loci of gene biotype “rRNA” or of non-mitochondrial RepeatMaskerloci of class “rRNA” (as defined in the RepeatMasker track retrievedfrom the UCSC Table Browser). FASTQ quality was assessed using FastQC(version 0.11.7), and alignment quality was assessed using RSeQC(version 3.0.0).

Variance-stabilizing transformation (VST) was accomplished using the“Variance Stabilizing Transformation” function in the DESeq2 R package(version 1.23.10) [55]. (Principal Component Analysis (PCA) wasperformed using the prcomp R function with variance stabilizingtransformed (VST) expression values that were z-normalized (set to amean of zero and a standard deviation of one) across all samples withineach gene. Differential expression was assessed using the likelihoodratio test and Wald test implemented in the DESeq2 R package. Correctionfor multiple hypothesis testing was accomplished using theBenjamini-Hochberg false discovery rate (FDR). All analyses wereperformed using the R environment for statistical computing (version4.0.2).

Statistical Analysis

To identify genes whose expression changes coordinately with respect toexosome treatment groups, a one-way analysis of variance (ANOVA) wasperformed using a likelihood ratio test to obtain a p value for eachgene. Benjamini-Hochberg False Discovery Rate (FDR) correction wasapplied to obtain FDR-corrected p values (q values), which represent theprobability that a given result is a false positive based on the overalldistribution of p values. Corrected/adjusted p values such as the FDR qare the best measure of significance for a given test when manyhypotheses (genes) are tested at once. The FDR q value was alsorecomputed after removing genes that did not pass the “independentfiltering” step in the DESeq2 package. Genes with low overall expressionare more strongly affected by random technical variation and more likelyto produce false positive results. Wald tests were then performed foreach gene between experimental groups to obtain a test statistic and pvalue for each gene. Statistical analyses of the in vitro experimentswere performed using Student's t test or ANOVA as indicated, and weregenerated by GraphPad Prism software. p<0.05 was consideredstatistically significant.

Results

Exosomes from Plasma of T2D Subjects Induce EMT in Prostate Cancer CellLines

Exosomes from conditioned media of mature adipocytes inducetranscription of EMT genes in breast cancer cell lines. This inductionis more pronounced if the adipocytes are insulin resistant or obtainedfrom the adipose tissue of subjects with T2D [49]. First, it was testedwhether plasma exosomes phenocopied this behavior, and induced EMT genesin prostate cancer cell lines. Exosomes were purified from EDTA-treated,anticoagulated peripheral blood plasma of three T2D subjects and treatedDU145 cells with equal numbers of exosomes (10⁹ in all cases) for twodays, then isolated total RNA and assayed expression of the classicalEMT genes vimentin (VIM), E-cadherin (CDH1), ANHAK and Snail (SNAI1) byRT-PCR with TaqMan probes. As hypothesized, T2D plasma exosomes inducedEMT genes compared to equal numbers of exosomes purified from plasma ofND subjects or media (complete growth media containing RPMI-1640 +10%FBS) control exosomes (FIG. 15A, FIGS. 21A-21B). Having thus validatedthe DU145 cell responses by RT-PCR, these same total RNAs were nextanalyzed on a commercial array for well-established EMT genes [49] andit was observed that the T2D plasma ex5osomes induced a coherent,pro-EMT signature in the DU145 cells compared to ND controls (FIG. 1C).Of the significantly differentially expressed genes in the commercialmicroarray, the T2D exosomes most strongly repressed CDH1, which encodesE-cadherin, as expected and as we have previously published for thisgene associated with maintenance of the epithelial program andopposition to the mesenchymal program [49].

Ingenuity pathway analysis (IPA) of disease and function based on FIG.15C revealed that plasma exosomes from T2D subjects strongly inducedtumor cell aggressive features, such as cell spreading, protrusions,metastasis, cell motility and invasion compared to plasma exosomes fromND subjects (FIG. 16A), while pathways related to cell death byapoptosis or necrosis were downregulated by T2D exosomes, similar towhat we have reported previously [49]. To confirm that T2D plasmaexosomes also induce mesenchymal features (increased perimeter andelongation, and reduced circularity) as previously reported [49],morphological parameters were analyzed using ImageJ. DU145 cells treatedwith T2D plasma exosomes showed increased elongation and perimeter, anddecreased circularity, compared to cells treated with ND plasma exosomesand control cells treated with media-only exosomes (FIGS. 16B-16C).

Exosomes from Plasma of T2D Subjects Induce PD-L1 Expression in DU145Prostate Cancer Cells

Gene signatures of EMT have been associated in several tumor types withimmune infiltrates that express interferon-gamma (IFN-γ)-induced genes,and correspondingly with elevated expression of immune checkpointproteins, such as PD-L1 [56-58]. The inventors explored whether, inaddition to inducing EMT networks, T2D plasma exosomes also upregulateexpression of immune checkpoint genes compared to ND plasma exosomes. Itwas found that plasma exosomes from T2D subjects did indeed upregulategenes that encode receptors important in cancer immunotherapy, includingCD274 (FIG. 17A), which encodes PD-L1, and CD155 (FIG. 17B), whichencodes the poliovirus receptor and is associated with resistance toimmune checkpoint therapy in several cancer types.

Next, the effect of T2D exosomes on DU145 cells was analyzed, using acommercial array focused on genes involved in inflammation and cancerimmune crosstalk. The T2D plasma exosomes induced a coherent,pro-inflammatory signature in DU145 cells compared to ND plasma exosomes(FIG. 17C). IPA analysis of the immune/inflammation microarray data fromFIG. 17C showed that T2D plasma exosomes strongly upregulated majorpathways associated with angiogenesis, immune dysfunction and tumorprogression, compared to plasma exosomes from ND subjects (FIG. 22).

MicroRNA Profiling of Plasma Exosomes

Our previous studies on payload differences between exosomes releasedinto conditioned media by mature adipocyte cultures from T2D vs NDsubjects used mass spectrometry and proteomics to show that severalproteins, particularly TSP5, were overrepresented in T2D exosomes [49].Functional studies then showed that recombinant TSP5 alone was able toinduce transcription of EMT gene signatures [49]. Therefore, a similarproteomics-based approach was used to investigate peptide differencesbetween T2D and ND exosomes purified from human plasma. However,proteomics profiling of three ND and three T2D exosome isolates revealedno distinct pattern of peptides that were differentially representedbetween the groups (data not shown).

It was considered that miRNAs might be differentially expressed insteadof proteins, and might encode functional activities that drive theobserved EMT and immune checkpoint gene expression. MicroRNA profiles ofplasma exosomes from T2D and ND subjects were compared by a humanserum/plasma miRNA PCR array and it was found that the metabolicdifferences associate with a different miRNA signature. The five miRNAsthat clustered as the most increased in T2D plasma exosomes compared toND plasma exosomes were: miR-374a-5p, miR-93-5p, miR-28-3p, miR532-5pand miR375 (FIG. 18A). Other miRNAs showed reduced differentialexpression in T2D plasma exosomes compared to ND plasma exosomes. Thefive miRNAs that clustered as the most decreased were: miR-326,miR424-5p, miR-27a-3p, miR320b and miR320d (FIG. 18A).

Three of FIG. 18A′s were obtained miRNAs from commercial sources andDU145 cells were treated with 25 nM of each, for 2 days. Then totalcellular RNA was isolated, cellular cDNA synthesized and fold-changes ofSNAI1 CD274 and CDH1 were compared to ACTB as measured by RT-PCR withTaqMan probes (FIGS. 18C-18D). It was found that miR374a-5p upregulatedCD274 (FIG. 18C) but not SNAI1 (FIG. 18B) and slightly downregulatedCDH1 (FIG. 18D); miR-93-5p upregulated SNAI1 (FIG. 18B) but not CD274(FIG. 18C) and did not affect CDH1 (FIG. 18D); and miR-424-5pdownregulated CDH1 (FIG. 18D) but had no effect on SNAI1 (FIG. 18B) orCD274 (FIG. 18C). These results supported the overall hypothesis thatT2D exosomal miRNAs are functionally active in tumor cell line models,when assayed as individual recombinant miRNAs.

A control experiment was also performed to prove that exosomal RNAs aretaken up by DU145 cells. Exosome RNA was stained with RNA selective dye,then the stained exosomes were added to DU145 cells and visualized byfluorescence microscopy over several hours to track localization. Timecourse analysis showed that after 16 and 24 hours, the plasma exosomesRNA became concentrated in the nuclei (FIGS. 23A-23B). Uptake reached aplateau by 16 hours.

RNA Sequencing and Principal Component Analysis (PCA)

Next, the global transcriptional changes in prostate cancer cell linescaused by T2D exosomes in comparison to the ND exosomes wasinvestigated. RNA sequencing and Principal Component Analysis of allsignals showed that total RNA transcripts from DU145 cells treated withexosomes isolated from plasma of ND subjects had similar globaltranscription patterns. The transcripts from cells treated withmedia-only control exosomes and ND plasma exosomes clustered togetherwell. However, transcripts from DU145 cells treated with exosomes fromT2D subjects were widely separated and unique. T2D samples 1 and 2clearly separate from the other samples along PC1 (24% of variance) andPC2 (14% of variance), respectively, indicating that there issignificant biological variability with respect to treatment with theT2D exosomes (FIG. 24A). The interpretation of this result is that NDplasma exosomes did not induce significant variation in genome-widepatterns of RNA expression compared to negative controls, but theexpression induced by different T2D patient exosomes differ widely fromND controls, each in their own way. Furthermore, when T2D plasmaexosomes are compared to ND, a group of genes upregulated specificallyby miR-103 (FIG. 24B) was identified immune exhaustion.

IPA analysis of differential expression of all the genes in the datasetswas undertaken. Network analysis revealed that miR103a and SOX2-OTinduce EMT and PD-L1 expression. MiR-103a and SOX2-OT were upregulated28.8 and 21.8 times in DU145 cells treated with T2D exosomes compared toND exosomes (Table 2, FIG. 24C). Interestingly, these two genes weredownregulated (5 and 3.6-fold, respectively) in the ND exosome groupcompared to the media-only exosome control group (Table 2). The RNA seqdatasets were further analyzed by IPA and the potential connections ofdysregulated genes and cancer cell EMT and immune exhaustion through BETproteins were explored. IPA analysis revealed that miR103a and SOX2-OTinduce Cav1 and VIM respectively, which ultimately promote EMT and PD-L1expression through BRD4 (FIG. 5). Additionally, RNA seq data showed thatSNAI1 and CAV1 had an upregulated trend although it was not significant(FIG. 24B).

The somatic BET bromodomain proteins BRD2, BRD3 and BRD4 play criticalroles in transcriptional control of classical EMT genes [53, 65, 66], aswell as control of immune checkpoint genes, such as PDCD1 and CD274,which encode PD-1 and PD-L1, respectively. These findings have sincebeen confirmed by others [67-69]. It was therefore hypothesized herethat the BET protein family functions as a central node inexosome-driven signal transduction, linking EMT to immune checkpointfunction, and demonstrated here in prostate cancer cells. In DU145 cellslow concentrations of the BRD4-selective PROTAC degrader MZ-1 (50 nM)are indeed selective to eliminate BRD4 protein while preserving BRD2 andBRD3, whereas high doses (400 nM) eliminate all three somatic BETproteins [51]. The small molecule JQ1 inhibits BET bromodomain activitythrough a different mechanism by competitively binding to the histonebinding pocket of the bromodomain, and is not highly selective amongBRD2, BRD3 and BRD4, thus it was used as a positive control forinhibition of all BET proteins, not just BRD4. The transcription ofSNAI1and CD274 genes was measured by RT-PCR. It was found that MZ-1 atBRD4-selective concentrations in DU145 cells inhibited expression ofSNAI1induced by T2D plasma exosomes, and the pan-BET inhibitor JQ1 wasnot able to reduce expression below the BRD4-selective level (FIG. 19A).Interestingly, MZ-1 had the same inhibitory effect on CD274, but JQ1 wasable to inhibit transcription to below baseline (FIG. 19A). It was thenverified by flow cytometry that PD-L1 protein expressed on the surfaceof treated DU145 cells was induced by T2D exosome treatment, andinhibited by MZ-1 or JQ1 (FIG. 19B).

Discussion

Prostate cancer exhibits significant genomic and histologicheterogeneity that complicates prognostic assessment and clinicaldecision making. Disease can be indolent and localized, oligoclonal withnon-overlapping mutational profiles among nearby clones [70], oraggressive with rapid progression and metastatic dissemination of alethal clone [71]. A large subgroup of cases appears to be indolent atthe early stage; it is important to resolve the indolent cases fromaggressive cases that demand immediate treatment. Although serum levelof Prostate Specific Antigen (PSA) has proven utility in combinationwith digital rectal examination for diagnostic screening [72, 73], PSAaccuracy is suboptimal to understand cancer risks [74, 75].

Described herein is a new approach of exploring plasma exosomes forfunctional biomarkers for, e.g., prostate cancer patients. This reportis the first to describe how plasma exosomes from subjects with Type 2diabetes (i) drive pro-EMT transcriptional shifts and (ii) elevateimmune checkpoint expression in human prostate cancer models.Surprisingly, it was also found that plasma exosomes from ND controlsshowed activity to downregulate certain classical, pro-EMT genes, suchas SNAI1 and to upregulate certain classical anti-EMT genes, such asCDH1, in prostate cancer cell lines (FIG. 15C). This observationindicates that ND status can encode chemopreventive factors that arepackaged into plasma exosomes that circulate and protect againstprostate cancer progression.

The initial approach to use differential proteomics analysis of theexosomes to identify peptides with significantly different abundancebetween T2D and ND exosomes recapitulated the previous approach, whereproteomics analysis of exosomes purified from conditioned media ofinsulin-resistant vs insulin-sensitive mature adipocytes was used [49].The inventors turned now instead to analysis of miRNAs to identifypotential differences. Here, it was found that commercial array kitswere adequate to reveal interesting miRNA profiles.

To investigate this model, the RNA seq data of DU145 cells treated withplasma exosomes from T2D and ND subjects were compared. We found thatthe T2D exosomes upregulated a subset of genes that play critical rolesin both EMT and immune exhaustion (Table 2). In order to illustrate thepathway, genes and their fold change values were imported to IPA. Byusing the path explorer feature of the software, the connections amongupregulated genes, EMT genes and immune exhaustion genes were revealed(FIG. 19A). IPA output revealed that miR103 and SOX-2-OT stimulate CAV1and VIM respectively. Downstream in the pathway, BRD4 acts as thecritical node, and activates SNAI1and CD274, which subsequently driveEMT and immune checkpoint expression. In addition to miR-103, SOX-2-OTwas significantly upregulated in DU145 cells treated with T2D plasmaexosomes.

Taken together, these data indicate a new signaling map to link thepro-EMT effect of T2D plasma exosomes with PD-L1 expression through BRD4(FIG. 20). PD-L1 targeting is a major treatment strategy for severalcurrent cancer clinical trials of checkpoint inhibitors. The PDL1findings have clinical relevance related to utility of checkpointinhibitors for treatment of advanced prostate cancer. Althoughcheckpoint inhibitors have demonstrated significant efficacy in otheradvanced malignancies, the outcomes in advanced prostate cancer havebeen disappointing. Randomized phase III studies have failed todemonstrate an overall survival benefit in advanced prostate cancer [81,82]. These studies, as well as several smaller studies with PD-1/PD-L1inhibitors, did demonstrate clinical benefit in a small percentage ofpatients, yet there is ongoing need to identify markers to predictresponse or resistance. As we show here, Type 2 diabetes works throughplasma exosome crosstalk to increase the plasticity of prostate cancerprimary cells, and induce immune exhaustion markers important formicroenvironment interactions with tumor-infiltrating T cells.

In conclusion, the present findings show that T2D plasma exosomes induceprominent EMT and immune checkpoint markers in DU145 cells. This studyis the first report to unravel the oncogenic function of T2D plasmaexosomes that drives metastatic progression and treatment failure incastration-resistant prostate cancer.

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TABLE 2 Symbol fold.change_ND_vs_Con fold.change_T2D_vs_Confold.change_T2D_vs_ND MIR103A2 −5.005253486 5.561461796 28.87518193ARL4AP5 −1.95414835 11.57645127 22.58555682 SOX2-OT −3.679618116.016827533 21.86199733 C7orf61 −16.19318846 1.287780561 21.09214409FXYD7 −15.97775953 1.194140851 18.89103921 HRC NA 18.3480239618.38664791 MIR1286 −6.042447073 3.004941052 17.62304769 PIN1-DT−11.61622709 1.505049851 17.27275791 LINC01635 −7.21885436 2.37795132617.21696202 EIF4BP9 NA 17.03338232 17.061045 VN1R84P −4.0251925624.407524333 16.93310321 MSANTD3-TMEFF1 −5.467967563 3.09312073716.84184086 MIR4668 −3.794992095 4.503584138 16.65817178 SOAT2−7.145222742 2.327222179 16.58960244 PMPCAP1 −7.375271802 2.25877282316.50219214 ACOT6 NA 16.39857397 16.41723447 RNFT1P3 −1.954148358.433102195 16.41723447 RPS15AP36 −5.364217591 3.039649782 16.28294562PSG7 −10.38224754 1.565430829 16.20783701 ENPP7P4 NA 16.1171570716.10324678 OSTCP6 −1.95414835 8.291342455 16.10324678 RPS26P58 NA15.28160126 15.31373522 GAPDHP2 NA 14.63451474 14.66541845 RNU6-1266P−1.95414835 7.498300224 14.66541845 RN7SL753P NA 14.51615745 14.53121252RPL23AP49 NA 14.51615745 14.53121252 XACT −5.364217591 2.69970099614.53121252 MIR6749 NA 14.34477297 14.37494052 CCT7P1 −5.2513568562.689326562 14.29752482 FEN1P1 −7.145222742 1.969050084 14.08447622MTND4P9 −9.048147608 1.554401104 14.0230489 MIR6895 NA 13.8170510113.81477337 APOC1P1 −7.039067573 1.94904635 13.72478559 CRMP1−7.360603914 1.882017812 13.72478559 NT5DC4 −7.258223528 1.90526654413.72478559 ATP5POP1 NA 13.64889395 13.66175235 DSCAS −22.93864992−1.648352108 13.66175235 SYS1-DBNDD2 NA 13.43514808 13.4236218 ZSCAN5BNA 13.43514808 13.4236218 MYCLP1 −3.576580763 3.398912283 12.24346321ZFP3-DT −3.338560543 3.581557246 12.24346321 MIR320E −5.3434004692.224704053 12.01099843 EVX1-AS −3.576580763 3.305025103 11.90637711RN7SL673P −1.95414835 6.051839571 11.83925105 C6orf47-AS1 −1.954148355.939793252 11.61391653 EEF1A1P17 NA 11.58951773 11.61391653 TNRC18P2 NA11.51790377 11.5344449 GCKR 1.941286913 22.43553484 11.53390578 CDH22−1.95414835 5.882701961 11.48067719 LINC01191 −1.95414835 5.8833068711.48067719 BOLA3P2 NA 11.24564302 11.2611859 ASPG −12.40052338−1.130515356 10.83595921 AMD1P3 −16.73045385 −1.923512558 8.786012607EBLN1 −9.710708538 −1.139082694 8.433357667 PON1 −10.14244961−1.206643366 8.433357667 YIPF7 1.941286913 15.28160126 7.835115047HNRNPA1P43 1.941286913 14.93286278 7.660002825 LYPLA1P2 1.94128691314.21937742 7.304044781 TRAPPC12-AS1 1.941286913 14.21937742 7.304044781SYT9 −10.69818306 −1.493394981 7.101129339 RASL11B 1.94128691313.52581433 6.960067237 RN7SL233P 1.941286913 13.43514808 6.90919633MAP4K1-AS1 1.93779033 13.04484309 6.861066418 KRT8P43 1.83776186212.14129508 6.567584828 MSLNL 1.941286913 12.21772748 6.259597984 NXF51.941286913 12.21772748 6.259597984 SLC25A18 −9.828509403 −1.633572216.112170834 TTLL7-IT1 −10.08339379 −1.667936375 6.112170834 CALB1−10.83767733 −1.88414567 5.804673018 GOLGA6A −12.16755032 −2.122451895.804673018 LINC02478 −14.18711158 −2.464381726 5.804673018 ITFG2-AS1−18.40603109 −3.224037325 5.738753 LINC00514 −18.09604507 −3.171212975.738753 LINC00839 3.532167601 19.90597601 5.566211957 DHRS9−12.71429102 −2.283182058 5.513617533 NR1I3 3.359444518 19.03170795.5049762 C8orf37-AS1 −14.30238364 −2.576952131 5.455394776 IL34−15.97775953 −2.877319162 5.455394776 MEF2C-AS1 −9.603789983−1.727468999 5.455394776 PCNAP3 −9.998729208 −1.822776479 5.455394776S100A5 −12.72644249 −2.289749512 5.455394776 NACAP2 −16.39604187−2.983523671 5.376040403 RPS2P35 −10.8231515 −1.968469606 5.376040403TFPI2-DT −13.95942298 −2.53758511 5.376040403 TUBB7P −16.50564813−2.991348858 5.376040403 RNU6ATAC27P 3.791983406 17.21142169 4.6031537RN7SL566P 3.791983406 16.86150174 4.510505684 MIR3188 3.74137699516.34118817 4.432166905 ST8SIA1 −9.810908439 −2.236021136 4.394075959PLA2G4D −9.862996446 −2.31895451 4.197116874 SNORA2C −12.79069411−2.993739578 4.196935597 TACR2 5.470052118 22.31792376 4.091404515PPM1B-DT 3.532167601 13.74537168 3.874795924 RPS29P11 3.7955638412.51549912 3.29540518 DUTP5 −10.08339379 −3.306039019 3.040969275 GREM2−11.1741458 −3.663983679 3.040969275 NCAN −10.61115995 −3.4848698093.040969275 TMEM151B −14.07215785 −4.617154809 3.040969275 TOX−13.18453787 −4.323685583 3.040969275 ZNF439 −9.603789983 −3.1469495273.040969275 LEF1-AS1 5.635914096 14.8730302 2.643906751 MIR39425.591091016 14.553007 2.612937938 PRB3 5.259436861 13.648893952.582360634 RNU2-11P 5.550542991 13.74537168 2.483664794 KRTAP5-107.441317315 18.34802396 2.454771828 ELANE 7.531635955 18.348023962.430015001 PSMC1P11 10.7288115 25.85980574 2.398148607 METTL1P16.94078147 16.34118817 2.339064348 TMEM178A 8.389108217 16.506726021.946265873 KRT18P24 7.401750341 13.88907825 1.881042995 UVRAG-DT8.958595731 16.64672223 1.856433685 FHAD1-AS1 14.38344291 25.301394781.756325906 RNU4-82P 6.895707273 12.21772748 1.743595235 DRICH1−9.983897406 −6.377333494 1.561316724 LINC02273 14.79695992 19.678071141.334022351 ADAM21P1 12.62327361 16.70293361 1.323803384 YWHAQP614.89179559 16.75663828 1.129885801 MTX1P1 12.94862208 14.516157451.11446628 RSPH10B 12.69723135 13.43514808 1.065830705 ODAD2P114.01227818 14.21937742 1.006010808 ANKH-DT 12.41012118 11.88122465−1.057560665 EXTL1 12.32880203 11.51790377 −1.082425387 KRTDAP13.12728448 11.82202401 −1.119909098 GAL3ST2 −7.795498866 −9.379773228−1.219461753 LINC02450 −10.84319839 −13.46400157 −1.247484692 THAP12P3−7.500532301 −9.343797493 −1.247484692 PCDHGA11 10.29376946 7.32344995−1.411400198 NCCRP1 12.32880203 8.775667058 −1.427497052 SNORD121A16.82912387 10.99844258 −1.523829498 IGLV1-50 13.28994553 8.066997329−1.640721741 PAPPA-AS1 16.48003044 9.156784442 −1.851273062 PFN1P1116.39356435 8.548018223 −1.927233361 ZCRB1P1 9.488113901 4.519391044−2.128697818 RNU4-5P 12.32880203 5.792244848 −2.15861817 LINC01121−3.631523652 −8.581952359 −2.380969581 SMAD5-AS1 −3.983680008−9.351822884 −2.380969581 FAM90A2P −4.303167361 −10.11949762−2.409022859 RPSAP3 −4.714392984 −11.09303601 −2.409022859 RPL7AP6014.79695992 5.501917716 −2.662711594 ERAS 15.21143917 5.38052126−2.777039494 GMFG −3.277470518 −10.92471505 −3.222913366 CASC1910.06387269 3.03751863 −3.318339706 SLC39A5 10.06387269 3.03751863−3.318339706 SCN3A −3.197069026 −11.04137182 −3.486347121 NOSTRIN10.64420431 3.03751863 −3.513115191 MIR3659HG −2.63209785 −9.194122085−3.515887642 ZNF365 −2.430730235 −8.757645292 −3.608425972 RN7SL605P11.10145328 3.03751863 −3.660794839 INHBC 11.88248381 3.108430533−3.954960211 UBASH3A 12.19592537 3.03751863 −4.022401431 ISLR212.27400704 3.03751863 −4.050165488 KRT18P3 12.491658 3.03751863−4.122092933 SNORA68B 12.78567296 3.03751863 −4.216986982 KRT18P18−2.299893975 −10.56092913 −4.566298419 HNRNPH1P1 −1.854577022−8.737087989 −4.675693853 MUC20P1 −3.226844632 −14.98418325 −4.675693853SMYD3-IT1 −1.919746307 −9.071964133 −4.766095576 CICP14 −2.156143973−10.19708749 −4.820127561 GUSBP17 −1.587224712 −8.749174049 −5.495483533CCDC185 −3.058191309 −17.47777522 −5.669375744 SSTR2 −1.565565318−8.947953204 −5.730208872 NTN5 −2.092939914 −12.10726294 −5.787207683RNU6-1 −1.888376929 −11.02220952 −5.812584626 DUSP5-DT −2.323326792−13.47776557 −5.86548236 HLA-DOA −1.9777014 −11.5557531 −5.893228675OR2AG2 −1.890209043 −11.11931356 −5.982977399 ATXN7L3-AS1 18.165551573.03751863 −5.99305182 ACTG1P16 −1.527078077 −9.06057004 −6.040926537EXOC3L1 9.428425544 NA −6.071659994 RPS10P2 9.428425544 NA −6.071659994RN7SL75P 10.06830058 1.559546761 −6.466979376 RAB25 10.42526375 NA−6.68612331 RPL22P4 10.76020833 NA −6.914896158 RN7SL663P −1.294658636−9.06057004 −7.083184173 RPL37P1 11.01184317 NA −7.083184173 TSSK3−1.932011554 −13.55080123 −7.111447117 GPR88 11.10520311 NA −7.150691197RPL11P1 11.10520311 NA −7.150691197 RPL7AP15 11.15715492 NA −7.178386137SLC6A15 11.15715492 NA −7.178386137 RPL23AP81 −1.643596918 −11.70415839−7.203169246 TMPRSS11D 11.18639398 NA −7.203169246 GNG8 11.27725201 NA−7.262285942 RNU6-1062P 11.38885747 NA −7.335147148 RPL21P65−1.182425279 −8.986309942 −7.510875049 CALCB −1.184718929 −9.282917726−7.800131787 SETP6 12.37260804 NA −7.941528882 LINC01277 −1.770096398−14.21765816 −8.105497114 SPCS2P1 12.62327361 NA −8.10898234 TPM3P6−1.356281155 −11.17330924 −8.162253747 NDST1-AS1 −1.344765413−10.87408353 −8.219506411 CCT6P4 12.77879724 NA −8.221175169 ZMYND12−1.249339962 −10.33818944 −8.361184201 PAK3 3.57138664 −2.353788054−8.395057488 GJD4 1.846270969 −4.485708299 −8.400479977 SH3TC2-DT2.430482143 −3.495319315 −8.559286963 RPL10P19 2.419344435 −3.879914307−9.066446301 DHX35-DT −1.263100885 −11.5557531 −9.131799631 RNU6-4P2.593768606 −3.552055812 −9.204909024 AK5 −1.084838923 −9.890003709−9.241867375 POC1B-GALNT4 7.406906609 −1.248901844 −9.269975236 S1PR114.42690634 NA −9.269975236 BMP3 14.45888151 NA −9.29635945 LINC020861.200064627 −7.607826418 −9.298428203 SNORD42B 1.115534388 −8.493655649−9.437199787 MMP13 1.306451369 −7.289472222 −9.475068756 ALDH8A11.195403699 −7.99866325 −9.518630381 FNDC7 7.589768112 −1.248901844−9.520429066 NXPH2 1.664837723 −5.633282003 −9.520429066 LINC017111.494652207 −6.304932453 −9.532751851 RNU6-1263P −1.088951774−10.2719814 −9.55008141 LINC02551 −1.866949313 −17.76312047 −9.615943029LGALSL-DT 1.393373391 −6.839605235 −9.623085508 LINC01238 14.97796178 NA−9.645338599 TBCEL-TECTA −1.084765814 −10.33818944 −9.645338599 PCDHGB8P1.40822853 −7.132656081 −9.909038433 MIXL1 1.010815775 −9.894591466−10.04498935 MAGED4B 1.237974472 −8.201607845 −10.06786169 CYP21A1P1.240807147 −8.437943433 −10.40516857 HSPD1P12 1.88259871 −5.402592792−10.43124127 RNU6-126P 4.46506153 −2.353788054 −10.43124127 GFI1B−1.22196946 −12.83941581 −10.54188293 CHRNA4 2.970098586 −3.642305209−10.65521794 RN7SL130P 4.803441744 −2.209341304 −10.92938747 PDCL3P62.02015341 −5.7745143 −11.70572992 FAM83E 1.310501579 −9.22803198−12.05848948 RPL39P5 −1.143100003 −14.37748314 −12.69993531 GCM22.840365945 −4.485708299 −12.94997958 LINC00167 1.103284269 −11.70415839−12.95575494 LINC01768 1.107680653 −11.69702861 −13.00610567 HNRNPA1P2710.6022635 −1.248901844 −13.31196668 TEN1 3.093222345 −4.3392147−13.8359405 MIR579 4.159637865 −3.350702681 −14.21596734 ZNF32-AS21.875726522 −7.985547968 −15.07910025 RIPOR3 4.360920581 −3.495319315−15.24592472 C17orf64 6.600643215 −2.353788054 −15.44877987 RAD21-AS12.336259348 −6.923825947 −16.24290064 RNU6-9 2.439001599 −7.132656081−17.23616482 PSMC1P3 2.189027097 −8.289942024 −18.00760246 PKN2-AS12.151501777 −9.06057004 −19.70123815 LINC01473 5.777058261 −3.350702681−19.8392474 SNAP23P1 2.288636611 −9.06057004 −20.97327262

1. A method comprising: determining the expression of at least one gene selected from the group consisting of: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, AHNAK, miR-424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b, and miR320d; in an exosome obtained from a subject.
 2. The method of claim 1, wherein the expression of at least one gene selected from the group consisting of: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, miR-424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b, and miR320d; is determined.
 3. The method of claim 1, wherein the expression of at least one gene selected from the group consisting of: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, and miR-375; is determined.
 4. The method of claim 1, wherein the expression of at least two genes selected from the group consisting of: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, and miR-375; is determined.
 5. The method of claim 1, wherein the expression of at least three genes selected from the group consisting of: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, and miR-375; is determined.
 6. The method of claim 1, wherein the expression of at least four genes selected from the group consisting of: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, and miR-375; is determined.
 7. The method of claim 1, wherein the expression of at least miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, and miR-375; is determined.
 8. The method of claim 1, wherein the expression of at least miR374a-5p is determined.
 9. The method of claim 1, further comprising: i) administering a glucose-controlling medication or obesity medication and/or ii) administering CT scans at a frequency of higher than 1 CT scan every 6 months, to a subject determined to have an expression level of at least one gene selected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, and AHNAK which is increased relative to a reference; or an expression level of at least one gene selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d; which is decreased relative to a reference.
 10. The method of claim 1, further comprising: i) administering a glucose-controlling medication or obesity medication and/or ii) administering CT scans at a frequency of higher than 1 CT scan every 6 months, to a subject determined to have an expression level of at least one gene selected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, and AHNAK which is increased relative to a reference; or an expression level of at least one gene selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d; which is decreased relative to a reference; or i) not administering a glucose-controlling medication or obesity medication and/or ii) administering CT scans at a frequency of no more than 1 CT scan every 6 months, to a subject determined to have an expression level of at least one gene selected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, and AHNAK which is not increased relative to a reference; or an expression level of at least one gene selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d; which is not decreased relative to a reference.
 11. A method of treating cancer, comprising: i) administering a glucose-controlling medication or obesity medication and/or ii) administering CT scans at a frequency of higher than 1 CT scan every 6 months, to a subject determined to have an expression level of at least one gene selected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, and AHNAK which is increased relative to a reference; or an expression level of at least one gene selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d; which is decreased relative to a reference.
 12. The method of claim 11, further comprising: i) not administering a glucose-controlling medication or obesity medication and/or ii) administering CT scans at a frequency of no more than 1 CT scan every 6 months, to a subject determined to have an expression level of at least one gene selected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, and AHNAK which is not increased relative to a reference; or an expression level of at least one gene selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d; which is not decreased relative to a reference.
 13. The method of claim 11, wherein the glucose-controlling medication is selected from the group consisting of: metformin, a sulfonylurea, a glinide, a SGLT2 inhibitor, and insulin; or the obesity medication selected from the group consisting of: orlistat, phentermine-topiramate, naltrexone-bupropion, liraglutide, semagludtide, setmelanotide, phentermine, benzphetamine, diethylpropion, and phendimetrazine.
 14. The method of claim 1, wherein the level of expression is the level of mRNA.
 15. The method of claim 1, wherein the exosome originates from or is isolated from a non-tumor tissue or cells.
 16. The method of claim 15, wherein the non-tumor tissue or cells is blood, plasma, adipose tissue, adipocytes, or bone.
 17. The method of claim 11, wherein the cancer is an epithelial cancer.
 18. The method of claim 17, wherein the cancer is an epithelial adenocarcinoma, esophageal cancer, pancreatic cancer, cervical cancer, colorectal cancer, gastric cancer, lung cancer, uterine caner, renal cancer, breast cancer, or prostate cancer.
 19. The method of claim 11, wherein the subject is diabetic, overweight, or obese.
 20. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject exosomes which are: a) from a non-diabetic and/or non-obese donor; and/or b) determined to have an level of expression of at least one gene selected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, and AHNAK; which is not increased; and/or a level of expression of at least one gene selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d; which is not increased, wherein the level of expression is relative to the level of expression in a exosome obtained from a healthy non-diabetic subject.
 21. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject: a) an inhibitor of at least one gene selected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, and AHNAK; which is not increased; and/or b) an agonist of at least one gene selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d;
 22. The method of claim 20, wherein the subject is one determined to have: an increased level of expression of at least one gene selected from: miR374a-5p, miR-93-5p, miR-28-3p, miR-let-7b-3p, miR-375, TSP5, Snail (SNAI1), Twist (TWIST1), Slug (SNAI2), vimentin (VIM), E-cadherin (CDH1), ZEB1, and AHNAK; or a decreased level of expression of at least one gene selected from: miR424-5p, miR-326, miR424-5p, miR-27a-3p, miR320b and miR320d. 